Rodent inflammasome activation by Toxoplasma gondii

by

Kimberly M. Cirelli

B.S./M.S. Molecular and Cellular BiologyJohns Hopkins University, 2010

SUBMITTED TO THE DEPARTMENT OF MICROBIOLOGY IN PARTIALOF THE REQUIREMENTS FOR THE DEGREE OF

ARCHIVESMASSACHUSETTS INSTITUTE

OF TECHNOWGY

JUL 052016

LIBRARIES

FULFILMENT

DOCTOR OF PHILOSOPHY IN MICROBIOLOGYAT THE

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

FEBRUARY 2016

C2016 Kimberly M. Cirelli. All rights reserved.

The author hereby grants to MIT permission to reproduce and to distribute publicly paper andelectronic copies of this thesis document in whole or in part in any medium now known or

hereafter created.

Signature of Author

Certified by:

.Signature redactedD

Signature redactedAss

epartment of MicrobiologyNovember 1 2015

Jeroen Saeijociate Professor of Biology

Thesis Supervisor

Accepted by: Signature redacted_____Kristala Jones Prather

Associate Professor of Chemical EngineeringCo-Director, Graduate Program in Microbiology

Rodent inflammasome activation by Toxoplasma gondii

by

Kimberly M. Cirelli

Submitted to the Department of Microbiology on November 19 th 2015 in Partial Fulfillment ofthe Requirements for the Degree of Doctor of Philosophy in Microbiology

Abstract

Toxoplasma gondii is an obligate intracellular pathogen capable of chronically infectingnearly all warm-blooded animals, including humans. The chronic stage is characterized by thepresence of semi-dormant cysts in brain and muscle tissues. These cysts are crucial in the successof Toxoplasma as they are orally infectious and allow for the transmission of the parasitebetween hosts. As the host immune response drives cyst formation, the establishment of thischronic infection relies on the parasite's ability to find a balance between activation of a hostimmune response and evasion of parasiticidal mechanisms. This balance is achieved through themodulation of host cell processes by parasite proteins secreted from specialized secretoryorganelles known as rhoptries and dense granules. Here, we report that Toxoplasma activates theinflammasomes in mice and rats. The inflammasomes are a set of cytoplasmic patternrecognition receptors (PRRs). Activation of the inflammasomes results in caspase-1 activationand the cleavage and release of the pro-inflammatory cytokines, Interleukin (IL)-1 and IL-18.IL-1p is an important mediator of local inflammation and neutrophil recruitment. IL- 18 inducesInterferon (IFN)-y, which is a critical cytokine in the control of Toxoplasma. A form of celldeath, termed pyroptosis, can accompany inflammasome activation.

The NLRP3 inflammasome is activated in mouse macrophages, leading to the secretionof IL-i I in vitro. The NLRPI and NLRP3 inflammasomes play a major role in mouse survivaland control of parasite replication in vivo. The NLRPI inflammasome is activated in infectedmacrophages from rats that are able to completely clear infection. Toxoplasma infection leads tothe secretion of active IL-I and IL-18. Activation of the NLRP1 inflammasome leads topyroptosis, a programmed form of cell death. Pyroptosis prevents parasite replication within thehost cell and likely promotes clearance by nearby immune cells. Using a chemical mutagenesisscreen, we identified three Toxoplasma dense granule proteins (GRAs), GRA18, GRA27 andGRA28, essential for NLRP1 inflammasome activation and pyroptosis in rat macrophages. Ourwork has identified Toxoplasma gondii as a novel activator of the rodent inflammasomes anddemonstrated host cell death as a mechanism to control parasite replication. We have alsoidentified three novel parasite proteins required for this activation, providing insight intointeractions between parasite and host, which may aid in the treatment of human infection.

Thesis Advisor: Jeroen SaeijTitle: Associate Professor of Biology

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Acknowledgements

First and most importantly, I want to thank my advisor and mentor, Jeroen. Without yoursupport and guidance, I would not have been able to accomplish this. I want to thank you forcreating such an incredible lab environment, for your endless patience, ideas and both yourprofessional and personal advice. I could not have asked for a more supportive mentor.

I'd like to thank my thesis committee members. To Dennis, thank you for your supportthroughout graduate school and it was an absolute pleasure to able to TA for you in 7.26. ToHidde, thank you for your feedback over the years and bringing fresh ideas to my project. I wantto thank Doug Golenbock for being on my defense committee.

The Saeij-entists are a group of amazing scientists and friends. Lindsay, you taught mehow to pass parasites, have a good attitude when an experiment fails and provided excellentShonda Rhimes commentary. Ana, thank you for making me laugh in tissue culture and bringingvino into my life. Mariane, thank you for your guidance at the beginning of this project andalways making lab a party. Musa, I will always appreciate your poor life advice early on Tuesdaymornings. Emily, you pushed me to think better scientifically and always made me laugh.Ninghan, your sassiness brought lightness to lab that can't be replicated. Natalle, thank you forbeing my student and helping me become a better teacher. Kirk, your immunological knowledgeleft me in awe and you showed me what it was like to be really moved by a publication. Dan,thank you for your advice about science and food. Ben, your enthusiasm and attitude have beenso refreshing these past two years. Eleni, Wendy, Quynh, Darlene, Judy, Joris, Renee, Diana,Deepshikha, Yaning, Lauren, Ken, Stephanie, Kiva, Monique, Brittany, thank you for bringinglight to the lab and for being great colleagues throughout graduate school. Our Boyer labneighbors have provided endless support throughout the years, particularly Vidya and Gizem.

I have made some great friends throughout graduate school and without their support, Icould not have stayed sane. I want to thank my roommate of four years, Simina, who has gonethrough every major step of graduate school next to me. In particular, I want to thank my bestfriend, Kenny. Thank you for supporting me through thick and thin and I can't wait to start thenext stage together.

Lastly, I want to thank my family. My parents have taught me that I can do anything anddid everything in their power to help make my dreams come true.

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Table of Contents

Abstract............................................................................................................................................3Acknow ledgem ents..........................................................................................................................5Table of Contents.............................................................................................................................7List of Abbreviations.......................................................................................................................9Chapter One: Introduction.............................................................................................................11

Toxoplasma gondii is a m odel intracellular pathogen.................................................. 12Disease m anifestation................................................................................................... 13Toxoplasma life cycle................................................................................................... 14Genetic diversity................................................................................................................16Activation of the immune system ................................................................................... 17Interferon-gamma induced immunity to Toxoplasma gondii....................................... 20The inflam m asomes...................................................................................................... 22

Rats are a better m odel of human toxoplasm osis........................................................... 25Toxoplasma effector proteins........................................................................................ 26Findings presented in this thesis................................................................................... 27References..........................................................................................................................31

Chapter Two: Dual role for inflammasome sensors NLRPI and NLRP3 in murine resistance toToxoplasma gondii.........................................................................................................................41

Abstract .............................................................................................................................. 42Importance.........................................................................................................................42Introduction........................................................................................................................43Results ................................................................................................................................. 46Discussion..........................................................................................................................60M aterials and M ethods................................................................................................... 67Supplementary Figures................................................................................................. 71Acknowledgements........................................................................................................ 74

References..........................................................................................................................75Chapter Three: Inflammasome sensor NLRP1 controls rat macrophage susceptibility to

Toxoplasma gondii.........................................................................................................................82Abstract..............................................................................................................................83Author Sum m ary................................................................................................................84Introduction........................................................................................................................85Results ................................................................................................................................ 88Discussion........................................................................................................................104M aterials and M ethods.....................................................................................................109Supplem entary Figures....................................................................................................116Acknowledgements..........................................................................................................124References........................................................................................................................125

Chapter Three: Addendum ........................................................................................................... 129Results and Discussion....................................................................................................130M aterials and M ethods.....................................................................................................135References........................................................................................................................137

Chapter Four: Three novel Toxoplasma gondii dense granule proteins are required for Lewis ratNLRPI activation.........................................................................................................................138

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Abstract ............................................................................................................................ 139Introduction ...................................................................................................................... 140Results .............................................................................................................................. 142Discussion ........................................................................................................................ 168M aterials and M ethods ..................................................................................................... 172Supplementary Figures .................................................................................................... 177Acknowledgements .......................................................................................................... 180References ........................................................................................................................ 181

Chapter Five: Conclusions and Future Directions ....................................................................... 185Summary .......................................................................................................................... 186Discussion and Future Directions .................................................................................... 187References ........................................................................................................................ 196

List of AbbreviationsAIM2 - Absent in Melanoma 2ASC - Apoptosis-associated Speck-like protein containing a CARDBBB - Blood-Brain BarrierBMDM - Bone Marrrow-Derived MacrophagesBN - Brown NorwayCARD - Caspase Activation and Recruitment DomainCas9 - CRISPR Associated Protein 9CRISPR - Clustered Regularly Interspaced Short Palindromic RepeatsDAMP - Damage-Associated Molecular PatternDC - Dendritic CellDS - Dextran SulfateGBP - Guanylate-Binding ProteinGO - Gene OntologyGOI - Gene of InterestGPI - GlycosylphosphatidylinositolGRA - Dense granule proteinGSDMD - Gasdermin DHFF - Human Foreskin FibroblastsHIV - Human Immunodeficiency VirusIDO - Indoleamine 2,3-dioxygenaseIFN - InterferonIg - ImmunoglobulinIL - InterleukiniNOS - Inducible Nitric Oxide SynthaseIPAF - Interleukin- 1 n-converting enzyme Protease-Activating FactorIRF - Interferon Regulatory FactorIRG - Immunity-Related Guanosine TriphosphatasesLD - Lethal DoseLEW - LewisLF - Lethal FactorLPS - LipopolysaccharideLRR - Leucine-Rich RepeatLT - Lethal ToxinMAPKK - Mitogen-Activated Protein Kinase KinaseMHC - Major Histocompatibility ComplexMIC - Micronemal proteinNAIP - NLR family, apoptosis inhibitory proteinNFKB - Nuclear Factor Kappa-light-chain-enhancer of activated B cellsNK - Natural Killer cellNLR - Nod-Like ReceptorNLRC - NLR family CARD domain-containing proteinNLRP - NACHT, LRR and PYD domains-containing ProteinNO - Nitric OxideNOD - Nucleotide-binding Oligomerization Domain

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PA - Protective AntigenPAMP - Pathogen-Associated Molecular PatternPRR - Pattern Recognition ReceptorPV - Parasitophorous Vacuole

PVM - Parasitophorous Vacuole MembranePYD - Pyrin domainRON - Rhoptry neck proteinROP - Rhoptry proteinSD - Sprague DawleySNP - Single Nucleotide PolymorphismTLR - Toll-Like ReceptorTNF - Tumor Necrosis Factor

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Chapter One:Introduction

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Toxoplasma gondii is a model intracellular pathogen

The World Health Organization has established infectious disease as the second leading

cause of death in the world, accounting for approximately 25% deaths (World Health

Organization, 2004). A majority of these infectious diseases are caused by intracellular

pathogens, including Mycobacterium tuberculosis, the causative agent of tuberculosis, Human

Immunodeficiency virus (HIV), Plasmodium, the causative agent of malaria and

Cryptosporidium, which causes the food-borne diarrheal disease cryptosporidiosis (McDonald et

al. 2013). Studying the interaction between host and pathogen is critical in the development of

more effective treatments. While bacteria and viruses have been long studied and many

mechanisms through which these pathogens cause disease have been elucidated, much less is

known about how eukaryotic pathogens cause disease.

Plasmodium, Eimeria, Neospora and Cryptosporidium are protozoan parasites and

members of the phylum Apicomplexa. Apicomplexan parasites can infect livestock and can

cause significant economical loss. Eimeria is a major pathogen in poultry (Chapman et al. 2013)

and Neospora is a major cause of abortions in cattle (Mazuz et al 2014). Because of their

clinical relevance, studying the host-parasite interactions of these organisms is especially

important. However, these organisms are relatively difficult to study. Several species of

Plasmodium cause human malaria, but only one has been successfully cultured in vitro.

Cryptosporidium has only been established to grow in culture recently and is difficult to

genetically engineer (Vinayak et al. 2015). Toxoplasma gondii, another apicomplexan parasite,

serves as an excellent model for these organisms. Toxoplasma is a pathogen of humans and

livestock, causing abortions in sheep and goats (Buxton 1990). Its genetic manipulability and

tractability and ease of use in in vitro and in vivo studies make Toxoplasma an incredibly useful

12

tool in dissecting parasite biology. Toxoplasma has been used to identify several host and

pathogen factors that determine disease outcome.

Disease manifestation

Toxoplasma gondil is an obligate intracellular parasite and a leading cause of human

death due to food-borne illness in the United States (Hoffmann, Batz, and Morris 2012).

Toxoplasma is capable of infecting all nucleated cells of all warm-blooded animals. Infection is

lifelong and chronic infection is characterized by the presence of semi-dormant cysts in the

muscle tissues and brain. It is estimated that one-third of the human population is chronically

infected with Toxoplasma (Sibley and Ajioka 2008). Infection rates vary by region, with an

estimated 10% of humans in the United States and 80% of those in Brazil chronically infected

(Pappas et al. 2009).

Infection is usually asymptomatic in immunocompetent individuals. In

immunocompromised individuals, such as HIV/AIDS patients, Toxoplasma is an important

opportunistic pathogen and can cause brain encephalitis. During the AIDS epidemic in the

1980s, Toxoplasma infection of the central nervous system was diagnosed in up to 20% of

patients (Velimirovic 1984). Additionally, Toxoplasma is a dangerous pathogen to developing

fetuses in newly infected pregnant women, as the parasite is able to cross the placenta and infect

the fetus. Toxoplasma is a leading cause of miscarriage and birth defects in humans (McLeod et

al. 2012). Infection can also result in ocular disease, even in immunocompetent individuals. In

Brazil, nearly 18% of examined individuals had ocular toxoplasmosis (Roberts 1999).

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Toxoplasma life cycle

While the range of hosts Toxoplasma is capable of infecting is vast, the definitive host

range is much narrower and limited to the members of the Felidae (feline) family. When a cat

ingests an infectious form of Toxoplasma, the haploid parasite undergoes differentiation in the

feline gut (Figure 1) (Sibley and Ajioka 2008). This differentiation into microgametes and

macrogametes allows the mating of the two and the formation of diploid oocysts. If two distinct

strains are simultaneously present, the parasites can undergo sexual recombination, resulting in

numerous FI progeny with a genome derived from both parental strains. The oocysts are shed in

the feline feces into the environment, where they will undergo sporulation, forming haploid

sporozoites (Dubey 2009). These oocysts are highly infectious and environmentally stable. Upon

ingestion of infectious Toxoplasma (either tissue cysts or oocysts) by an intermediate host, host

acids, including pepsins, digest the cyst wall, releasing the parasite. Toxoplasma sporozoites or

bradyzoites released from the cysts will initially invade intestinal epithelial cells. There they will

convert into tachyzoites that can cross the intestinal epithelium, in which macrophages and

dendritic cells are the predominant cell type initially infected (Mordue and Sibley 2003; Suzuki

et al. 2005).

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Definitive host: Catdr

Graz in g

Carnivorism

Oocyst

Carn vorism MeiosisTachyzoites

convertto bradyzoitesGraz in g Sporocysts

Intermediatehosts ngerital

Figure 1. Life cycle of Toxoplasma gondii. Meiosis and sexual recombination can only occur infelines, which are Toxoplasma's definitive hosts. The oocysts are shed into the environment.Ingestion of contaminated water or food by intermediate hosts leads to acute infection. When ahost mounts an immune response, the tachyzoites convert into semi-dormant encystedbradyzoites in the brain and muscles, establishing a lifelong chronic infection. An intermediatehost can ingest infected intermediate host tissues, allowing for horizontal transfer. Adapted from(Sibley and Ajioka, 2008)

The tachyzoite can infect a wealth of host cells and disseminate throughout the body of

the host. Upon infection of a nucleated cell, the tachyzoite forms a non-fusogenic

parasitophorous vacuole (PV), in which it can replicate. After several rounds of replication, the

parasites will egress, lysing the host cell and the tachyzoites are free to invade new host cells.

The establishment of a chronic infection is key to the success of the parasite. Toxoplasma

must be able to migrate from the site of infection (the gut) to distal sites, including the brain.

Several models have been proposed to explain how the parasite is able to relocate and cross the

Blood-Brain Barrier (BBB): (1) free tachyzoites actively cross barriers between individual host

15

cells (paramigration) (Dobrowolski and Sibley 1996), (2) tachyzoites may invade one part of a

host cell and exit through the other side (paracellular migration) (Morisaki, Heuser, and Sibley

1995) and (3) the "Trojan horse" model, in which Toxoplasma exploits the trafficking ability of

leukocytes (Courret et al. 2006; Lambert et al. 2006; Lambert and Barragan 2010).

When the host mounts an immune response to this acute infection, tachyzoites will

convert into the slowly growing bradyzoite form. This stage of infection is characterized by the

presence of the tissue cysts in nervous and muscular tissues and remains throughout the life of

the host. This chronic stage of infection is critical for the success of the parasite as it is the only

orally infectious stage other than the oocyst. Asexual transmission of the parasite occurs through

the ingestion of infected hosts by other intermediate hosts.

Genetic diversity

Many different Toxoplasma strains have been isolated from a range of hosts throughout

the world. In Europe and North America, Toxoplasma clonal lineages, known as types I, II and

III dominate (Howe and Sibley 1995). Although these strains are genetically highly similar, they

differ in specific phenotypes such as virulence in mice, growth in vitro and cytokine induction

(Saeij, Boyle, and Boothroyd 2005). For instance, type I strains are virulent in mice (Lethal Dose

(LD)100 = 1), while type II and type III strains are less virulent (LD5o = 103 and 10 5, respectively)

(Sibley & Boothroyd 1992). The uses of sexual crosses between strains that differ in virulence

and quantitative trait locus mapping have identified specific parasite genes as modulators of host

signaling and immune pathways.

South America is home to many genetically diverse Toxoplasma strains. Non-type I, II or

III strains, termed atypical strains, can cause ocular toxoplasmosis and more severe disease than

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the clonal lineages (Lehmann et al. 2006; Bossi et al. 2002). Genome-wide single nucleotide

polymorphism (SNP) analysis of 26 strains, including South American strains, determined there

is significant diversity between these strains (Minot et al. 2012). Another SNP analysis sorted

Toxoplasma strains into 14 haplogroups (Su et al. 2012). The geographic regions where

haplogroups are isolated are distinct. While types II and III are found predominately in Europe

and Africa (Dubey et al. 2006; Al-Kappany et al. 2010; Herrmann et al. 2013), they are rarely

isolated in South America (More et al. 2012). In North America, types II, III and 12 are most

commonly found (Khan et al. 2011). Types 4, 5, 6, 8, 9 and 14 are found in South America (Su et

al. 2012). This geographic diversity and differences between strains are likely due to adaptations

of different strains to different hosts with distinct immune systems.

Activation of the immune system

The immune system is divided into two arms: innate and adaptive. The innate immune

system is the first line of defense and critical in the recognition and initial response against

pathogens. Adaptive immunity is responsible for immunological memory and upon re-exposure

to a pathogen, leads to a faster and stronger response to eliminate the invading organism. In order

for Toxoplasma to successfully establish a chronic infection, it must keep the host alive and

therefore must be recognized by the innate immune system so a host response is mounted, but

the parasite must be able to modulate this response so it is not entirely cleared by the host.

Throughout its lifetime, a host is challenged by a barrage of bacteria, viruses and

parasites. In order to recognize the presence of a wide variety of these pathogens, hosts have

evolved to sense conserved, often essential structures, found in many pathogens that are

otherwise very different. These structures, pathogen-associated molecular patterns (PAMPs) are

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sensed by pattern recognition receptors (PRRs). There are several families of PRRs that have

been well characterized and studied in the context of Toxoplasma infection.

Toll-like receptors (TLRs) recognize a variety of ligands present on bacteria, viruses and

parasites. Activation of the TLRs leads to a signal cascade that culminates in the activation of

signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells

(NF-KB) and interferon regulatory factors (IRFs). The NF-KB pathway regulates gene expression

in a number of host cellular processes, including cell proliferation and the production of

inflammatory cytokines, including interleukin (IL)-12 (Hayden and Ghosh 2008). IRFs are

transcription factors whose target genes include type I interferons (IFNs), which are involved in

an anti-viral response.

In the murine model of toxoplasmosis, TLR1 1 and TLR12 are primarily expressed in

dendritic cells (DCs) and recognize the Toxoplasma essential protein, profilin (Yarovinsky et al.

2005; Koblansky et al. 2013; Raetz et al. 2013) (Figure 2). Several models have been proposed

regarding the cooperation of TLR1 1 and TLR12 to recognize profilin in mice. It is suggested

profilin recognition by TLR12 homodimers is sufficient to initiate an immune response

(Koblansky et al. 2013), but an alternative model suggests a TLR1 1/TLR12 heterodimer is

necessary for this response (Andrade et al. 2013; Raetz et al. 2013). In humans, TLR11 is a

pseudogene and TLR12 is absent from the genome, suggesting other innate pathways are

activated by Toxoplasma in humans.

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Macrophage or dendritic cell

IRF8 ---

TLR11/12

Profilin

Any nucleate

IFNy

d cell Effector Mechanisms:

Immune Related GTPases

Nitric OxideReactive Oxygen Species

Figure 2. Activation of the TLRs by Toxoplasma gondii. In macrophages and primarily dendritic cells,

the Toxoplasma actin-binding protein, profilin, is recognized by the endosomal TLRI I and TLR 12.

Activation of TLRI 1/12 leads to the translocation of the transcription factor, IRF8 into the nucleus and

the expression of several host genes, including IL-12. IL-12 stimulates T cells and NK cells to secrete

IFN-y, which acts on several cell types. IFN-y upregulates the expression of the immune-related GTPases

and the production of nitric oxide and reactive oxygen species, which aid in parasite clearance (black

inhibitor). Toxoplasma is equipped with several parasite effectors to counter these host mechanisms

(green inhibitor)

TLR2 and TLR4 sense Toxoplasma glycosylphosphatidylinositols (GPIs) (Debierre-

Grockiego et al. 2007). TLR7 and TLR9 recognize parasite RNA and DNA respectively

(Andrade et al. 2013; Melo et al. 2010). TLR3/7/8/9/11/12 reside in endosomal compartments.

Localization of these TLRs to the endosomes is controlled by unc-93 homologue BI (UNC93B I)

(Kim et al. 2008; Pifer et al. 2011). Mice individually deficient in TLR3, TLR7, TLR9 or TLR1 I

are not more susceptible to infection than wild-type mice nor are triple TLR3/TLR7/TLR9

knockout mice. Mice deficient in UNC93BI are hypersensitive to infection with Toxoplasma

(Melo et al. 2010) as are TLR3/TLR7/TLR9/TLRI I quadruple knockout mice (Andrade et al.

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T cell L- 12NK cell

2013). This suggests the combined action of nucleic acid-sensing TLRs and TLR1 1 and TLR12

are required for full murine resistance to Toxoplasma.

Activation of the TLRs leads to the secretion of IL-12 by DCs and macrophages. IL-12

stimulates T cells and Natural Killer (NK) cells to produce Interferon (IFN)-y. IL-IP, together

with IL-12, has been found to stimulate the production of IFN-y in NK cells (Hunter et al. 1995).

IL-18 and IL-12 synergistically induce IFN-y expression in T cells (Okamura et al. 1998). IL-1I

and IL-18 are produced by myeloid cells through another set of PRRs, known as the

inflammasomes.

Toxoplasma-infected TLR1 1-deficient mice have significantly higher frequency and total

numbers of IFN-y+ cells than wild-type mice. These cells were not T cells or NK cells and were

determined to be neutrophils. Additionally, this IFN-y production is TLR-independent as

neutrophils from UNC93B 1-deficient mice produce greater levels of IFN-y than those of wild-

type mice. IL-i IP and Tumor Necrosis Factor (TNF), most likely from circulating monocytes and

resident macrophages, regulate this IFN-y production by neutrophils (Sturge et al. 2013). Human

neutrophils produce IFN-y (Ethuin et al. 2004). As humans do not express functional TLRI 1 or

TLR12, neutrophil-derived IFN-y may play a major role in human resistance to Toxoplasma. As

IL-lP is activated by the inflammasomes and is a major regulator of IFN-y production by

neutrophils, it is likely that inflammasome activation by Toxoplasma plays an important role in

humans.

Interferon-gamma induced immunity to Toxoplasma gondii

IFN-y is a crucial cytokine in the host response to Toxoplasma (Suzuki et al. 1988). Mice

deficient in IL-12 or IFN-y succumb to acute infection by avirulent parasite strains (Gazzinelli et

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al.; Scharton-Kersten et al. 1996). IFN-y upregulates genes encoding Major Histocompatibility

Complex (MHC) molecules, allowing greater antigen presentation (Zhou 2009; Steimle et al.

1994; Yang et al. 1995). IFN-y can also activate a wealth of cell types to mount parasiticidal

effector mechanisms. IFN-y induces the expression of the immunity-related GTPases (IRGs) and

p65 guanylate binding proteins (GBPs), which play an important role in murine defense against

Toxoplasma. The IRGs accumulate on the PVM and promote the destruction of the vacuole

(Taylor et al. 2004). The parasite is released into the cytosol, where the parasite can be destroyed

by lysosome-mediated degradation (Ling et al. 2006). Mice deficient in Irgml, Irgm3 or Igtp are

acutely susceptible to parasite infection (Taylor et al. 2000; Collazo et al. 2001). GBPs are

required for the recruitment of the IRGs to the PV (Yamamoto et al. 2012).

Additional host mechanisms mediated by IFN-y include the production of nitric oxide

(NO), which can inhibit parasite metabolic enzymes (Fang 2004; Adams et al. 1990).

Toxoplasma is an arginine auxotroph and recovers the amino acid from the host cytosol.

Inducible nitric oxide synthase (iNOS) utilizes L-arginine to produce NO, leading to the

reduction of available arginine in the cell, which restricts parasite growth (Fox, Gigley, and Bzik

2004). In addition to these parasiticidal mechanisms, NO serves as a signal to induce bradyzoite

conversion (Bohne, Heesemann, and Gross 1994). IFN-y also induces indoleamine-2,3 -

dioxygenase (IDO), which converts tryptophan into N-formylkynurenine. Tryptophan

auxotrophy renders Toxoplasma susceptible to this pathway, as a reduction in host tryptophan

suppresses parasite replication (Pfefferkorn, Eckel, and Rebhun 1986). Reactive oxygen species

also play a role in parasite control in mouse macrophages and human monocytes (Murray and

Cohn 1979; Murray et al. 1979).

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The inflammasomes

The Nucleotide-binding Oligomerization Domain (NOD)-Like Receptor (NLR) family

are a set of germ line-encoded cytosolic PRRs, which contains 22 human members and 34

murine members (Proell et al. 2008; Bryant and Monie 2012). Members of the family share a

leucine-rich repeat (LRR) region and a NACHT nucleotide-binding domain. NLR members can

be divided according to their N-terminal region. Members of the NLRP group contain an N-

terminal pyrin domain (PYD). NOD members contain caspase activation and recruitment

domains (CARD). IPAF members also contain a CARD domain, but are distinct from NOD

members. Proteins containing CARD are able to directly bind caspase proteins. PYD-containing

proteins are unable to bind caspases directly and require a scaffold protein, apoptosis-associated

speck-like protein containing a CARD (ASC or Pycard), which contains both PYD and CARD

domains.

Several members of the NLR family, including NACHT, LRR and PYD domains-

containing Protein (NLRP) 1, NLRP3 and NLR family CARD domain-containing protein

(NLRC) 4, act as sensors that are capable of forming macromolecular complexes known as the

canonical inflammasomes. Additionally, a member of the PYHIN protein family, absent in

melanoma 2 (AIM2) has been identified to assemble an inflammasome. Inflammasome sensors

vary in the types of ligands they recognize and their modes of activation. Despite differences in

mechanisms of activation, the sensors follow the same general pathway (Figure 4). Upon

recognition of a ligand, the sensors oligomerize into a large, 700kDa multimeric complex

(Martinon, Bums, and Tschopp 2002). Inactive caspase-1 is recruited to this complex, in which

the zymogen is cleaved. Now active caspase-I is able to cleave two of its substrates, pro-IL-1s

and pro-IL-18. In myeloid cells, pro-IL-18 is constitutively expressed (Puren, Fantuzzi, and

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Dinarello 1999). Pro-lL-P expression must be induced through the activation of the

transcription factor, NK-KB. A TLR agonist, typically the TLR4 agonist, lipopolysaccharide

(LPS), is used to induce pro-IL-1p expression in a step known as "priming" (Guo, Callaway, and

Ting 2015). Cleavage of these cytokines leads to their activation and release. IL-i p is involved in

local inflammation, recruitment of neutrophils to site of infection and the induction of IFN-y

production by neutrophils and natural killer cells (Dinarello 1996; Hunter, Chizzonite, and

Remington 1995). IL-18 acts on natural killer (NK) and T-cells, which release IFN-y (Dinarello

1998).

Cytoplasm

IL-1 pSensor IL-18

Inactive caspase-1

* *Agonistpro-IL-1Ppro-IL-18 Active caspase-1

Figure 3. Inflammasome activation. The inflammasomes are primarily expressed in myeloid

cells. Upon recognition of an agonist, several sensors oligomerize. The multimeric complex

recruits caspase-1, which is cleaved and activated. Caspase-1 cleaves pro-IL-18 and pro-IL-I ,

leading to their activation and secretion.

Caspase-1 activation can also be accompanied by a programmed form of cell death,

termed pyroptosis (Fink and Cookson 2005). In several gram-negative bacterial infections, a

non-canonical inflammasome containing another pro-inflammatory caspase, caspase-1 1, plays a

23

major role in host resistance. Caspase- 11 is activated by direct recognition of cytoplasmic LPS

(Shi et al. 2014). Caspase- 11 activation leads to caspase- 1-dependent IL-1p release and caspase-

1 1-dependent pyroptosis (Kayagaki et al. 2011). Very recently, gasdermin D (GSDMD) was

identified as a caspase- 1 and caspase- 11 substrate, whose cleavage was necessary and sufficient

to induce pyroptosis (Shi et al. 2015; Kayagaki et al. 2015).

Pyroptosis results in the swelling and lysis of the cell, releasing intracellular contents

(Chen et al. 1996; Hersh et al. 1999; Hilbi 1998; Hilbi et al. 1997; Brennan and Cookson 2000).

These contents include Damage-Associated Molecular Pattern Molecules (DAMPs), which can

activate nearby cells to mount an immune response. Because of this, pyroptosis is a pro-

inflammatory form of cell death (Cookson and Brennan 2001). Additionally, pyroptosis has been

associated with the control of intracellular microbes. Macrophages infected with intracellular

bacteria, including Salmonella, Legionella and Burkholderia, undergo pyroptosis, leading to the

release of the bacterium, preventing intracellular replication and exposing the microbe to

phagocytic cells, particularly neutrophils (Miao et al. 2010).

Allelic differences in human NALPJ, which encodes for the NLRP1 sensor, has been

linked to differences in susceptibility to congenital toxoplasmosis (Witola et al. 2011). The only

identified activator of the rodent NLRP1 inflammasome is lethal toxin (LT) produced by

Bacillus anthracis. Lethal toxin is composed of two proteins: lethal factor (LF) and protective

antigen (PA). PA is required for entry of the toxin into the host cytosol (Milne et al. 2006), while

LF is a zinc-dependent protease, which cleaves the N-terminus of mitogen-activated protein

kinase kinases (MAPKKs), inhibiting their activity (Vitale et al. 1998; Pellizzari et al. 1999;

Duesbery 1998). In anthrax-susceptible strains of rats, LT is also able to cleave the N-terminus

of NLRPI. This cleavage is necessary and sufficient to activate the inflammasome (Levinsohn et

24

al. 2012). In anthrax-susceptible and resistant mice, LT cleaves NLRPlb, suggesting there are

additional requirements for mouse NLRP1 inflammasome activation (Hellmich et al. 2012).

The P2X7 Receptor (P2X7R) is expressed on the surface of macrophages and activated

by extracellular ATP, often derived from damaged or dead cells. Macrophages infected with

Toxoplasma are able to clear infection when activated with ATP (Lees et al. 2010). Parasite

death is accompanied by host cell death. P2X7R activation leads to NLRP3 inflammasome

activation through the efflux of K+, although the precise mechanism of NLRP3 activation is not

fully understood (Petrilli et al. 2007).

Rats are a better model of human toxoplasmosis

As the host range of Toxoplasma is wide, there are relative differences in the

susceptibility of host species to the parasite. In addition, there are differences between different

strains within a species. The most commonly used host model for Toxoplasma is the laboratory

mouse, as it is an excellent tool to study host resistance and immunology. However, the mouse

may not be the most accurate model for human toxoplasmosis. Toxoplasmosis is generally

asymptomatic in immunocompetent humans. In contrast, immunocompetent mice are relatively

susceptible to infection. During acute infection, mice experience symptoms such as weight loss,

a hunched posture and extreme lethargy. Rats are an understudied model of toxoplasmosis, but

model human infection more accurately. Rats that are infected with high doses of mouse-virulent

parasites not only survive infection but also do not display the common symptoms of acute

infection seen in the mouse. Rats develop a chronic, asymptomatic infection.

Interestingly, a rat strain, Lewis, was identified to be completely resistant to Toxoplasma.

Regardless of parasite strain, dosage and route of infection, Lewis rats are able to clear infection

25

entirely. Toxoplasma failed to encyst in Lewis brains and muscle tissues (Kempf et al. 1999;

Sergent et al. 2005). Additionally, Lewis rats had significantly lower titers of anti-Toxoplasma

antibodies. Neutralization of IFN-y led to an increase in antibody titer, but failed to allow

formation of tissue cysts (Sergent et al. 2005). Neutralization of IFN-y in control rats led to an

increase in Toxoplasma-specific antibodies and brain cysts. This suggest that IFN-y plays a role

in rat control of infection, likely through parasite replication, but Lewis rats utilize another

mechanism to clear infection.

Using bone marrow chimera studies, the resistance of Lewis rats was found to be intrinsic

to bone marrow-derived cells. Additionally, through the use of crosses between Lewis rats and

susceptible rats, the resistance was found to be a dominant trait (Sergent et al. 2005). Utilizing

F2 progeny and recombinant inbred rats, the resistance was mapped to a single 1.3cM locus on

chromosome 10, termed Toxo] (Cavailles et al. 2006). Toxol includes approximately 250

annotated rat genes. Contained within the locus is NlrpJ, encoding for the NLRP1

inflammasome sensor.

Toxoplasma effector proteins

Toxoplasma is equipped with three secretory organelles that are critical in establishing an

infection: the micronemes, rhoptries and dense granules. Micronemal (MICs) and rhoptry

proteins (ROPs) are primarily secreted during invasion (Figure 4). Upon recognition of a host

cell, MICs and rhoptry neck (RONs) proteins mediate attachment and invasion by forming a

moving junction, a structure through which the parasite pulls itself (Boothroyd and Dubremetz

2008). Rhoptry bulb proteins and some dense granule (GRAs) proteins are injected into the cell

upon invasion (Boothroyd and Dubremetz 2008; Rosowski et al. 2011). These proteins can

26

traffic to the PVM or to host cell locations, including the nucleus. The majority of GRAs are

secreted from the parasite once the PV has been established and continue to be secreted during

replication (Dubremetz et al. 1993).

ROPs and GRAs have been established as important parasite modulators of the host

response. ROP16 from type I and III strains acts as a tyrosine kinase that directly phosphorylates

and activates the host transcription factors, STAT3 and STAT6 and therefore affects host gene

expression (Saeij et al. 2007; Ong, Reese, and Boothroyd 2010). Certain allelic combinations of

ROP18 and ROP5, a kinase and pseudokinase respectively, counter the activity of the IRGs.

GRA15 from type II parasite strains is able to activate the transcription factor, NF-KB, which

leads to the expression of a wealth of host proteins, including proinflammatory cytokines (e.g.

IL-12 and IL- P) (Rosowski et al. 2011). GRA16 and GRA24 are secreted out of the PV and

traffic to the host nucleus, where they change host gene expression. GRA16 binds PP2A

phosphatase and the deubiquitinase HAUSP to positively regulate the tumor suppressor p53

(Bougdour et al. 2013). GRA24 interacts with p38a MAP kinase, inducing p38a's

autophosphorylation and activation. p38a activation correlates with increased expression of

several transcription factors including, Egr-1 and c-Fos (Bougdour et al. 2014).

27

Dense granules (GRAs)

00 0.

* / Micronemes (MICs)Rhoptries (ROPs)

pJ*0 Moving Junction

ROP18

Parasitophorous Vacuole GRA1 5Membrane (PVM)

Parasitophorous 00 ROP5Vacuole (PV) P

G GRA24*GRA16 ROP16S Host Nucleus

Figure 4. Toxoplasma specialized secretory organelles. Rhoptry neck and bulb proteins(ROPs, green) and micronemal proteins (MICs, purple) are secreted during invasion. Densegranule proteins (GRAs, red) are secreted constitutively during parasite replication.

Findings presented in this thesis

In chapter II of this thesis, we explored the role the inflammasomes play in murine

resistance to Toxoplasma infection. We determined that infected bone marrow-derived

macrophages (BMDMs) prepared from mice secrete active IL- I3, but do not undergo pyroptosis.

Using BMDMs from mice deficient in individual inflammasome components, NLRP3 was found

to be the predominant inflammasome activated by the parasite. BMDMs deficient in NLRPI did

not show a defect in IL-1 P secretion. In vivo, both NLRPI and NLRP3 played a role in mouse

resistance to Toxoplasma. Mice individually deficient in these sensors succumbed to infection

earlier than their wild-type counterparts with significantly higher parasite burdens. Additionally,

28

dOW

while systemic IL-i IP could not be detected, IL- 18 was found to be a critical cytokine in the

murine response to the parasite, most likely by inducing IFN-y.

In chapter III, we noted that the Lewis rat Toxoplasma resistance locus (Toxol) contained

the Nlrpi gene. We found that BMDMs isolated from the Toxoplasma-resistant Lewis rat, but

not susceptible Sprague-Dawley (SD) rat, infected with Toxoplasma undergo pyroptosis and

release active IL-1p and IL-18. Host cell death is a mechanism to prevent parasite replication, as

the majority of Lewis BMDMs contain single parasites after 24 hours of infection while

Toxoplasma replicates uninhibited in SD macrophages. A survey of Toxoplasma strains

representing worldwide diversity found that all strains tested were able to induce pyroptosis in

Lewis macrophages. Using several methods, we found Nlrpi required for inflammasome and

pyroptosis activation by Toxoplasma. Expression of Lewis Nlrpl in rat macrophages that do not

undergo pyroptosis was sufficient to sensitize the cells to infection-induced pyroptosis. Chapter

II and III identify Toxoplasma as the second activator of rodent NLRP 1.

In chapter IV, we designed a chemical mutagenesis screen where we isolated parasite

strains unable to induce pyroptosis in Lewis rat BMDMs. Using whole genome sequencing and

RNAseq, we identified the parasite genes mutated. Utilizing CRISPR/Cas9, we knocked out

several candidate genes and identified three novel dense granule proteins, GRA18, GRA27 and

GRA28 that are individually required for inflammasome activation. Parasites deficient in

GRA18, GRA27 or GRA28 fail to induce pyroptosis in Lewis BMDMs, replicate within the

Lewis macrophage and activate the Lewis inflammasomes significantly less than wild-type

parasites as measured by the release of active IL-i IP. The mechanism through which GRA 18,

GRA27 and GRA28 coordinate to activate NLRP1 is still unknown.

29

We have demonstrated the inflammasomes are an important innate immune pathway in

the control of Toxoplasma gondii and identified three novel parasites genes required for NLRP 1

activation. In Chapter V, we discuss how these findings have furthered our understanding of

parasite-host interactions and future directions.

30

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40

Chapter Two:Dual role for inflammasome sensors NLRP1 and NLRP3 in murine

resistance to Toxoplasma gondii

Gezahegn Gorfu*, Kimberly M. Cirelli*, Mariane B. Melo, Katrin Mayer-Barber, Devorah

Crown, Beverly H. Koller, Seth Masters, Alan Sher, Stephen H. Leppla, Mahtab Moayeri, Jeroen

P.J. Saeij and Michael E. Grigg

*Authors contributed equally to the paper.

Address correspondence to Mahtab Moayeri, mmoayeri@niaid.nih.gov; Jeroen P.J. Saeij,

jsaeij@ucdavis.edu; or Michael E. Grigg, griggm@niaid.nih.gov.

Kimberly Cirelli contributed to experiments in Figure 1A-D, 1G-N, S, S2 and S3. Gezahegn

Gorfu, Katrin Mayer-Barber and Devorah Crown contributed to experiments in Figures 2, 3, 4

and 5. Mariane B. Melo contributed to experiments in Figure lE and IF.

Originally published in mBio 5(1). February 18, 2014

41

Abstract

Induction of immunity that limits Toxoplasma gondii infection in mice is critically

dependent on the activation of the innate immune response. In this study, we investigated the

role of cytoplasmic nucleotide-binding domain and leucine-rich repeat containing a pyrin domain

(NLRP) inflammasome sensors during acute toxoplasmosis in mice. We show that in vitro

Toxoplasma infection of murine bone marrow-derived macrophages activates the NLRP3

inflammasome, resulting in the rapid production and cleavage of interleukin- 1 P (IL- 13), with no

measurable cleavage of IL- 18 and no pyroptosis. Paradoxically, Toxoplasma-infected mice

produced large quantities of IL- 18 but had no measurable IL-1p in their serum. Infection of mice

deficient in NLRP3, caspase-1/11, IL-IR, or the inflammasome adaptor protein ASC led to

decreased levels of circulating IL-18, increased parasite replication, and death. Interestingly,

mice deficient in NLRPI also displayed increased parasite loads and acute mortality. Using mice

deficient in IL-18 and IL-18R, we show that this cytokine plays an important role in limiting

parasite replication to promote murine survival. Our findings reveal T. gondii as a novel activator

of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these sensors in host

resistance to toxoplasmosis.

Importance

Inflammasomes are multiprotein complexes that are a major component of the innate

immune system. They contain "sensor" proteins that are responsible for detecting various

microbial and environmental danger signals and function by activating caspase-1, an enzyme that

mediates cleavage and release of the pro-inflammatory cytokines interleukin- 1 (IL-i IP) and IL-

18. Toxoplasma gondii is a highly successful protozoan parasite capable of infecting a wide

42

range of host species that have variable levels of resistance. We report here that T. gondii is a

novel activator of the NLRP1 and NLRP3 inflammasomes in vivo and establish a role for these

sensors in host resistance to toxoplasmosis. Using mice deficient in IL- 18 and IL-i 8R, we show

that the IL-18 cytokine plays a pivotal role by limiting parasite replication to promote murine

survival.

Introduction

The innate immune response plays a critical role in protecting hosts against pathogens.

Activation of innate immunity occurs after pattern recognition "sensor" proteins such as the Toll-

like receptors (TLRs) or nucleotide-binding domain and leucine-rich repeat-containing (NLR)

proteins detect the presence of pathogens, their products, or the danger signals that they induce

during active infection (Lamkanfi and Dixit 2012; Song and Lee 2012). Toxoplasma gondii is an

intracellular protozoan parasite capable of potently activating innate immunity in the wide range

of vertebrate species that it infects (Hunter and Sibley 2012; Melo, Jensen, and Saeij 2011). In

mice, resistance to T. gondii infection is critically dependent on the TLR-associated adaptor

protein MyD88, which is required for the induction of protective levels of the proinflammatory

cytokines interleukin-12 (IL-12) and gamma interferon (IFN-y) and the synthesis of nitric oxide

(NO) (Khan et al. 1997; LaRosa et al. 2008; Scanga et al. 2002; Scharton-Kersten et al. 1996;

Scharton-Kersten 1997; Sher et al. 2003; Suzuki et al. 1988). The activation and recruitment of

inflammatory monocytes to sites of infection are protective, as infection of mice rendered

deficient in Grl+ inflammatory monocytes by antibody depletion results in increased

susceptibility to parasite infection (Dunay et al. 2008; Robben et al. 2005). Furthermore,

chemokine receptor CCR2- and MCP1 (CCL2)-knockout (KO) mice, defective in recruitment of

43

these cells, are also more susceptible (Dunay et al. 2008; Robben et al. 2005). Hence, induction

of protective immunity against this protozoan pathogen is critically dependent on monocyte and

macrophage cell activation.

Macrophages are activated when their cognate receptors detect the presence of microbial

products. In the case of cytosolic NLRs, which sense the presence of microbes and/or the

damage that their infection induces, activation leads to the assembly of the inflammasome, a

multiprotein complex that recruits and activates caspase-1 and/or caspase-11. The murine

NLRP3 inflammasome senses a wide range of bacteria, pore-forming toxins, and crystalline

danger signals, including alum, amyloid clusters, cholesterol, and asbestos (Martinon, Mayor,

and Tschopp 2009). In contrast, the murine NLRPIb inflammasome is more restricted; the only

characterized activator is the Bacillus anthracis lethal toxin (LT) (Boyden and Dietrich 2006).

Either multimeric complex is capable of cleaving the proform of caspase-1, which is typically

associated with the rapid death of macrophages, through a process known as pyroptosis

(Lamkanfi and Dixit 2012; Song and Lee 2012). Pyroptosis, unlike apoptosis, leads to lysis of

the cell and release of its intracellular contents. Caspase-1 also cleaves the proinflammatory

cytokines IL-i I3 and IL-18, allowing their secretion from cells (Lamkanfi and Dixit 2012; Song

and Lee 2012). Whether the inflammasome is activated during Toxoplasma infection, or is

capable of altering disease pathogenesis, has thus far been only inferred. An association of

polymorphisms in the human Nlrpi gene with susceptibility to congenital toxoplasmosis was

recently reported (Jamieson et al. 2010; Witola et al. 2011). T. gondii production of cleaved IL-

1p in human monocytes is dependent on both caspase- 1 and the NLRP3 adaptor protein ASC

(Gov et al. 2013). P2X(7) receptors, which are important in ATP-mediated activation of the

NLRP3 inflammasome, have also been shown to influence parasite proliferation in human and

44

murine cells (Lees et al. 2010). IL- 18, a key substrate of inflammasome-activated caspase- 1, is

known to enhance production of IFN-y (Dinarello et al. 1998), which is a central regulator

of Toxoplasma pathogenesis. Furthermore, in vivo administration of IL-i IP protects mice from

lethal challenge with Toxoplasma (Chang, Grau, and Pechere 1990) and injection of antibodies

against the IL-I receptor (IL-i R) significantly attenuates the protective effect that exogenous IL-

12 confers on infected SCID mice (Hunter, Chizzonite, and Remington 1995) . Thus, we

hypothesized that inflammasome activation might be an important factor mediating murine host

resistance to Toxoplasma infection.

In this study, we show that murine macrophages are not susceptible

to Toxoplasma gondii-induced rapid pyroptosis but that NLRP3 inflammasome activation in

these cells results in rapid IL-ip cleavage and release. We establish that both NLRP3 and

NLRP1 are important in vivo regulators of parasite proliferation and that IL-18 signaling is

required to mediate host resistance to acute toxoplasmosis. Our findings establish a role for two

inflammasomes in the control of Toxoplasma infection.

45

Results and Discussion

Toxoplasma activates the inflammasome in murine macrophages without inducing cell

death

Induction of protective immunity capable of controlling murine Toxoplasma infection is

critically dependent on myeloid cell activation (Hunter and Sibley 2012). The ability of this

parasite to promote caspase-1 activation and the secretion of active IL-Ip has recently been

established in human and rat monocytes and macrophages (Witola et al. 2011; Gov et al. 2013;

Cirelli et al. 2014). To determine if Toxoplasma activates the inflammasome in murine

macrophages, we infected unstimulated and lipopolysaccharide (LPS)-primed bone marrow-

derived macrophages (BMDMs) prepared from C57BL/6J mice with type II (Pru) parasites and

measured IL-i IP secretion 24 hours after infection (Figure 1A). Uninfected BMDMs did not

produce measurable levels of IL-i IP (Figure 1A), whereas IL-i IP was readily detected after

infection with type II Toxoplasma regardless of whether the BMDMs were LPS primed or not

(Figure 1A). Western blotting of infected BMDM lysates showed the presence of mature IL-1p

and showed that cleavage was dependent on caspase-1/1 1, since infected BMDMs from caspase-

1/11-deficient mice did not possess detectable levels of cleaved IL-lP (Figure 1B). The

detection of mature IL-lP in the Toxoplasma-infected BMDMs indicated inflammasome

activation, but the cells did not undergo pyroptosis over 24 hours (Figure 1C). These data

support an inflammasome-mediated processing and release of mature IL-Ip in the absence of

pyroptosis, which have been demonstrated to occur previously (Broz et al. 2010). Interestingly,

IL-18 upregulation and cleavage were not observed in Toxoplasma-infected BMDMs or

46

splenocyte lysates over a range of multiplicities of infection (MOIs) and times, and the cytokine

was not released from in vitro-infected macrophages or splenocytes (data not shown).

Because IL-i IP secretion was consistently dependent on MOI (data not shown), we tested

whether parasite invasion was required for IL- I P secretion. Parasites pretreated with mycalolide

B, an actin-depolymerizing agent that blocks invasion but allows for secretion of microneme and

rhoptry contents, attached but induced significantly smaller amounts of IL-1 secretion

(Figure 1D), indicating that macrophage inflammasome activation was invasion-dependent. The

small amount of IL-10 secretion by BMDMs infected by mycalolide B-treated parasites was

likely due to incomplete inhibition of invasion, as immunofluorescence microscopy performed

on the same batch of treated parasites indicated that a small number had still invaded the

BMDMs, as evidenced by their intracellular replication (data not shown).

47

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Figure 1. Toxoplasma activates the inflammasome in C57BL/6 and 129S BMDMs. BMDMs wereprimed with 100 ng/ml LPS or left unstinulated for 2 hours and subsequently infected with type Iparasites (Pru; average MOL, 1) for 24 hours. (A) Quantification of IL- I P in supernatants was performed

48

I

- 17kD

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B

Rr

using ELISA. (B) IL-1p cleavage was monitored by Western blotting of cell lysates from C57BL/6NTacor caspase-1/1 1' BMDMs that were infected with type II parasites (Pru; MOI, 0.8) for 24 hours. Thepositions of both pro-IL-ip (37 kDa) and cleaved IL-1 (17 kDa) are indicated. (C) Cell viability ofinfected cells in panel A was determined at different time points using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Panels A and C are averagesof three experiments. Error bars, + standard deviations. (D) BMDMs were primed with LPS for 2 hoursand infected for 24 hours with type I parasites (RH) that were pretreated with dimethyl sulfoxide vehicleor mycalolide B (3 pM) for 20 minutes. IL-1p was measured using ELISA. Data are averages of 2experiments. Error bars, + standard deviations. (E) C57BL/6 BMDMs were infected with the indicatedstrains for 24 hours. IL-11 was measured using ELISA. Data are the averages of at least 3 experimentsper strain. The haplogroup to which the strain belongs is indicated above. Error bars, + standarddeviations. (F) C57BL/6 BMDMs were infected for 18 hours with Pru (type II) or PruAGRA15 (MOI, 4)for 18 hours, and microarrays were used to determine the fold change in IL- 13 mRNA expression levelscompared to uninfected macrophages. (G) BMDMs were infected with Pru (type II) or PruAGRA15 for24 hours. IL-11 was measured using ELISA. Data are representative of 3 experiments. Error bars, +standard deviations. (H) BMDMs were primed for 2 hours with LPS and then infected with indicatedstrains for 24 hours (MOI, 4). The haplogroup to which the strain belongs is indicated above. Data are theaverages of 3 experiments. Error bars, + standard deviations. (I) IL-13 secretion from primedimmortalized murine WT and caspase-1/ 1-' macrophages, infected for 24 hours with RH. Data shownare from an experiment representative of three. Error bars, + standard deviations. (J) IL- 11 secretion fromBMDMs prepared from wild-type C57BL/6 mice (blue) or C57BL/6 mice lacking Nlrpib (red), Nlrp3(green), Nlrpib and Nlrp3 (orange), or Asc (yellow), primed for 4 hours, and then infected with type I(RH; average MOI, 1) for 24 hours. Cytokine secretion below the detection level is indicated on the graphwith arrowheads and labeled not detected (n.d.). Data are averages of four experiments. Error bars, +standard deviations. (K) BMDMs described for panel J were primed for 3 hours with LPS and theninfected with type II parasites (MOI, 1.5) for 24 hours. Western blot analysis on concentratedsupernatants (25-fold), probing for cleaved IL-1p (17 kDa). (L) Host cell viability in panel L wasmeasured using the MTS assay. Error bars, + standard deviations. (M) BMDMs from C57BL/6 and 129Smice were primed with 100 ng/ml LPS for 2 hours and subsequently infected with type II parasites (Pru;average MOI, 0.7) for 24 hours. Quantification of IL-11 in supernatants was performed using ELISA.Panels L and M are the averages of 3 experiments. (N) 129S BMDMs were primed for 2 hours andinfected with type II parasites (MOI, 1.6) for 24 hours. Western blot analysis on concentratedsupernatants (25-fold), probing for cleaved IL-1p (17 kDa). Nig, nigericin; Tg, T gondii.

IL-1p secretion correlates with strain differences in NF-KB activation

Mouse strains differ in their susceptibility to Toxoplasma depending on the infecting

strain genotype; haplogroup 2 and 12 (HG2 and HG12) strains are relatively avirulent and

readily establish chronic infections, whereas HG1 and HG4 to HG1O strains are acutely virulent.

We sought to determine whether Toxoplasma strains differentially activate the murine

macrophage inflammasome, or whether secretion of IL-1p correlated with parasite genotype

49

and/or pathogenesis. We infected unprimed BMDMs from C57BL/6J mice

with Toxoplasma tachyzoites from all 12 haplogroups for 24 hours, a time point at which

parasite-induced cell lysis was minimal. Cougar (HG11) and the type II strains, with the

exception of DEG, induced IL-i IP secretion in unstimulated BMDMs (Figure 1E).

Inflammasome activation is often divided into a signal 1, which is the signal that leads to

transcription of I1-1/, and signal 2, which is the signal that leads to the actual activation of

caspase-1. Type II, but not type I or III, parasites directly activate the NF-xB transcription factor

in both human and murine cells, thereby potentially providing signal I for the induction of R1-

1,8 transcription. The secreted dense granule protein GRA15 determines this strain difference in

NF-KB activation (Rosowski et al. 2011). Indeed, in murine BMDMs, type II IL-1i/ mRNA

induction was partially dependent on type II GRA15 expression (Figure 1F), while IL-1p

secretion of unstimulated BMDMs was completely dependent on GRA15 (Figure 1G). To

determine if non-type II strains can provide signal 2, which leads to the activation of caspase-1

and subsequent cleavage and secretion of IL-1p, we prestimulated BMDMs with LPS for 2 hours

and subsequently infected them with different Toxoplasma strains. IL-i IP was now detected in

the medium with no truly apparent differences between strains (Figure 1H). The IL-1p secreted

into the medium also contained the cleaved active IL-1P (17 kDa) as determined by Western blot

analysis (Figure S2).

50

Toxoplasma activation of the murine inflammasome in BMDMs is dependent on caspase-

1/11 and NLRP3

To determine the components necessary for IL-1p secretion, we infected immortalized

macrophages that lacked caspase- 1 and -11 and showed that IL-pI P secretion was completely

eliminated, as expected (Figure 1I). To determine the inflammasome components necessary for

IL-1p secretion, we infected BMDMs from C57BL/6 mice that lacked Nirp1b, Nlrp3, or

both Nlrpib and Nlrp3, or the inflammasome adaptor ASC. We found IL-ip secretion by primed

BMDMs upon Toxoplasma type I infection to be mostly dependent on ASC and the NLRP3

inflammasome (Figure 1J). Similar results were obtained after type II infection (Figure S3).

The greatly reduced amount of cleaved active IL-i IP in the supernatant of Nlrp3-

deficient Toxoplasma-infected BMDMs compared to the amount present in the supernatant of

wild-type (WT) or Nlrplb-deficient infected BMDMs confirmed the importance of the NLRP3

inflammasome in Toxoplasma-mediated inflammasome activation in vitro (Figure 1K).

Thus, Toxoplasma induction of IL-i IP secretion by murine BMDMs is highly dependent on the

NLRP3 inflammasome and requires caspase- 1 activation.

Inflammasome-mediated BMDM death and cytokine processing are independent

of Nirpla and Nirpib alleles

Despite the activation of caspase-1 in Toxoplasma-infected cells, we did not observe any

macrophage pyroptosis. Previous reports have shown that five polymorphic Nlrplb alleles exist

among inbred mice which control sensitivity to anthrax LT-induced pyroptosis (Boyden and

Dietrich 2006). To test if the >100-amino-acid (aa) differences in Nlrplb between C57BL/6J and

129S mice were the basis for resistance to parasite-induced pyroptosis, we compared BMDMs

51

from the two strains. We observed no difference in cell viability between the C57BL/6J and

129S BMDMs (Figure 1L). Thus, consistent with a major role for NLRP3 inflammasome

activation, BMDMs from either 129S or C57BL/6 mice produced active IL-1p (Figure IM and

N) without associated pyroptosis upon type I or II Toxoplasma strain infection. Furthermore, the

fact that 129S BMDMs do not express the highly conserved NLRPla protein (Sastalla et al.

2013) and are caspase-1 1 deficient (Kayagaki et al. 2011) but present a robust IL-1IP response

also eliminated a role for NLRPla and caspase-11 in cytokine maturation induced

by Toxoplasma.

Murine resistance to Toxoplasma infection is controlled by caspase-1/11-dependent

inflammasome activation

Whether inflammasome activation is important to murine resistance to infection in vivo has

not yet been established. We infected mice deleted for the caspase-1/1 1 genes with 10,000 type

II 76K green fluorescent protein-luciferase (GFP-LUC) tachyzoites intraperitoneally (i.p.) and

tested for susceptibility to acute infection by monitoring mean survival time (MST), parasite

growth, dissemination, and the production of IL- 1p and IL- 18. In the absence of caspase- 1/11

proteins, mice had a 10- to 20-fold-higher parasite load (Figure 2A and B) and were highly

susceptible to acute infection (Figure 2C). In contrast, the majority of C57/BL6NTac control

mice survived acute infection and established chronic infections (Figure 2C). Surprisingly,

serum levels of systemic IL- I P never exceeded 10 pg/ml on day 5 (Figure 2D, graph on left) or

day 9 (data not shown) for either mouse strain. IL-18 levels, however, were significantly higher

following infection, ranging from 0.5 to 2.0 ng/ml in C57BL/6NTac mice on day 5 (Figure 2D),

52

to strikingly high levels exceeding 10 ng/ml by day 9, compared to <200 pg/ml in caspase-l/1 1-

deficient mice (Figure 2D) or uninfected controls (data not shown).

C

0i2:,

C57BL/6N CASP1/11--

1.83E+05 2.84E+06

3 69E+05 4.70E+06

2.96E+05 6.31 E+06

.- C57BL/6N

- Casp1/11-/-

106-

105-

y P6 n

Days Post Wnection

100-

50-

0

-+- C57BL/6N+Casp1/11-

I I I I

10 20 30 40

Days Post Infection

D2000-

1500-

1000-

500-

0*

2000-

1500-

.S1000-

500-

0

0000

0

000

Qk&m

Mop_,

0-'5'

(pC,

Figure 2. Parasite load, survival, and systemic IL-18 levels in caspase-I/1 I-deficient mice. (A)Bioluminescence imaging (BLI) of infected caspase-l/l I knockouts and controls on days 5 to 7 followinginfection with 76K GFP-LUC (10,000 tachyzoites i.p.). Images shown are for 3 mice. (B) Quantificationsare from 8 mice imaged/group. (C) Aggregate survival of caspase-1/l I-knockout mice (n = 13/group)compared with WT C57BL/6NTac control mice (n = 10/group). (D) IL-18 measurements in serum ofcaspase-I/l 1-deficient mice on day 5 after infection were significantly different from WT (P < 0.001)when infected with 76K GFP-LUC. No detectable levels of IL-fI3 were detected in circulation.

53

I1

A

B

ASC and NLRP3 inflammasome activation controls Toxoplasma proliferation and host

resistance

We next investigated the role of ASC in murine susceptibility to Toxoplasma infection,

since this adaptor protein is required to mediate the activation of multiple inflammasomes (Latz,

Xiao, and Stutz 2013). If inflammasome activation controls Toxoplasma resistance, we

hypothesized that mice rendered deficient in this protein will be more susceptible to acute

infection. Asc-deficient mice consistently had -20-fold or greater parasite loads at days 5 to 7

postinfection (Figure 3A and B) and generally succumbed to infection by days 8 to 10

(Figure 3C), in contrast to wild-type control mice, the majority of which survived acute

infection at day 20 (Figure 3C). The Asc-deficient mice likewise failed to induce detectable

levels of systemic IL- 18 (Figure 3D) or IL-1p (data not shown) during acute infection.

To determine whether the NLRP3 inflammasome sensor is sufficient to confer this

murine resistance to the Toxoplasma phenotype, Nlrp3' mice on the C57BL/6J background

were infected intraperitoneally with 10,000 76K GFP-LUC tachyzoites. Nlrp3' mice were more

susceptible than their wild-type (WT) controls and possessed 10-fold or higher parasite burdens

at days 5 to 7, and the majority of mice died by day 10 postinfection (Figure 3C). These infected

mice produced intermediate levels of systemic IL- 18, significantly greater than those of the Asc-

deficient mice but not equivalent to those of the WT (Figure 3D). They did not produce

measurable levels of IL- 1p (data not shown), similar to what was observed with all infections of

WT mice (Figure 2D and data not shown). Infection with another type II strain (Pru GFP-LUC)

produced similar results (data not shown), indicating that the phenotype was not attributable to

an anomalous Toxoplasma clone-specific effect or the genome integration site of the GFP-LUC

gene, as both type II strains activated the inflammasome in vivo. These results indicate that

54

murine resistance to acute infection with Toxoplasma is highly dependent on the activation of the

NLRP3 inflammasome.

A C

C57BL/6J ASC'-

2.74E+05 2.85E+06

100-N1I.rp30

1.06E+06

a,

C.

--_

3.43E+05 1.05E+07 4.36E+06

4,

12:

1 01-

106.

-1- C57BL6J-& ASC4--dr- NIrp34-

50-

I, II III

0

2000-

-- C57BL/6J-W- ASC-/--A- Nlrp3-/-

10 20 30 40

Days Post Infection

D

000

0

0

0

1500-

E

-a

5004 0

0

0

Days Post Infection7

01 :WC, P . e

Figure 3. Parasite load, survival, and systemic IL-18 levels in ASC- and NLRP3-knockout micefollowing Toxoplasma challenge. (A) Bioluminescence imaging of 76K GFP-LUC-infected mice(various strains, 10,000 tachyzoites, i.p. route) on days 5 to 7 following infection is shown. Images shownare from two or three representative mice from 2 to 6 mice/group from one representative experiment.The experiment shown is one of 3 (WT) or 2 (ASC and NLRP3) independent experiments. (B) P values(I test) comparing luciferase activity for each knockout strain to C57BL/6J are <0.05. (C) Aggregatesurvival curve of ASC (n = 8)- and NLRP3 (n = 5)-knockout mice compared with WT C57BL/6J controlmice (n = 13). (D) IL-18 measurements in serum of deficient mice on day 5 after infection. No detectablelevels of IL-1P were detected in circulation.

55

I__

B

I

NLRP1 inflammasome activation also controls Toxoplasma proliferation and host

resistance

Because the infected Nlrp3-deficient mice still produced IL- 18 at levels higher than those

of Asc-deficient mice, we hypothesized that more than one inflammasome is activated in vivo to

produce IL- 18 during Toxoplasma infection. To test this prediction, we infected mice deficient at

the Nlrpi locus encompassing Nlrplabc with 10,000 76K GFP-LUC tachyzoites to determine if

NLRPI activation contributes to murine resistance during Toxoplasma infection. Nlrplabc-

deficient mice had only 3- to 5-fold-higher parasite loads than did WT mice across days 5 to 7

(Figure 4A and 4B). Nlrplabc' mice also died acutely, succumbing to infection between days

16 and 22 (Figure 4C). The delayed MST kinetics was consistent with the decreased parasite

burden in comparison to the Nlrp3-deficient mice. Nlrplabc-deficient mice produced

intermediate levels of systemic IL- 18, significantly greater than those of the Asc-deficient mice

but not equivalent to those of the WT (Figure 4D). No measurable levels of circulating IL-Ip

were found on day 5 or 9 after infection in these mice or their WT controls (data not shown).

56

C57BL/6N

C

(.n

1.83E+05

100-

5.91E+05In

A

B

50-

- C57BL/6N-U- Nirp1-/--w

i io 20 30 40

Days Post Infection

2000-

1500-

Q 1000.

-00-

0.

0

00 00

00000(h00-

0

D

Figure 4. Parasite load, survival, and systemic IL-18 levels in NLRPI-knockout mice following

Toxoplasma challenge. (A) Bioluminescence imaging of 76K GFP-LUC-infected mice (various strains,

10,000 tachyzoites, i.p. route) on days 5 to 7 following infection is shown. Images shown are three

representative mice from 4 to 6 mice/group from one representative experiment. The experiment shown is

one of two WT or two NLRPI independent experiments. (B) P values (I test) comparing luciferase

activity for each knockout strain to C57BL/6NTac are <0.05. (C) Aggregate survival curve of NLRPI (n

= 10)-knockout mice compared with WT C57BL/6NTac control mice (n = 10). (D) IL-18 measurements

in serum of deficient mice on day 5 after infection. No detectable levels of IL-1p were detected in

circulation.

57

- - m -=11 mummlmt13.69E+05 1.21E+06

2.96E+05 2.09E+06

-0- C57BLrN

- Nirpi'10y-

104-

S 7

Days Post Ifection,

a I

Nirp1-4

9%

Mice deficient in signaling and secretion of IL-18 are highly susceptible to

Toxoplasma infection

Although no systemic IL-iP was detectable in vivo, infection of NLRP3-deficient BMDMs

showed markedly diminished levels of IL-i IP (Figure 1). To test if IL-1p possesses a biologically

important role locally and is capable of influencing murine resistance, we infected IL-I R-' mice

with 10,000 76K GFP-LUC tachyzoites. All IL-IR-deficient mice succumbed to infection but

with a delayed kinetics compared to ASC-, caspase-1/1 1-, and Nlrp3-deficient mice (Figure 5C).

These mice supported higher parasite loads and had intermediate levels of IL- 18 in their serum

compared to WT C57BL6/J mice (Figure 5A, B, and D), indicating that despite the absence of

measurable IL-ip in circulation, IL-i signaling does play a contributing role in the protection

against murine toxoplasmosis.

Intriguingly, the level of IL-18 in the circulation of infected WT mice correlated with

decreased parasite burden and increased survival. We hypothesized that inflammasome

activation and the production of high systemic IL- 18 might play an important role in the relative

resistance of WT mice. To test our hypothesis, we infected IL- 18/ and IL- I8R7' mice. Both

types of mice were highly susceptible to acute Toxoplasma infection and consistently had 20- to

>100-fold-increased parasite loads at days 5 to 7 postinfection, greater than that found in the

Asc-- mice (Figure 5A and B). These mice typically succumbed to infection by day 8 or 9

(Figure 5C), suggesting that the presence of IL-18 is protective in C57BL/6 mice. These data

indicate that the production and secretion of activated IL-18 are associated with controlling

parasite proliferation and murine resistance to acute toxoplasmosis.

58

A C57BL/6J IL-1R-'

1.47E+05 4.60E+05

6.36E+O5 1 59E+07

5.74E+05 5.05E+06

2.13E+05 2. 55E+07

B

-J

T

108-

107-

10.

105-

IL-184 IL-1 8R1

1.48E+06 4.65E+05

1.02E+07 2.31E+07

-RO6.15E+07 4.03E+07

9.43E+07 1.01 E+07

+- C57BLrJ. IL-18-/.-*- IL-18R-/--W- ILA-1--

Days Post Infection

C

16)

0CL

-40- C57BL/-6- IL-18-

1001 -V- IL-18R-+ IL-1 R-/-

50-

JL-V

D 2000-

150

E

CL 100

so

7

6J

0 10 20 30 40

Days Post Infection

000

0

0

0-

0- 0 0 000

00

00

0 q I

Figure 5. Parasite load, survival, and systemic IL-18 levels in IL-18- and IL-18R-knockout mice.

(A) Bioluminescence imaging of 76K GFP-LUC-infected mice (various strains, 10,000 tachyzoites, i.p.

route) on days 4 to 7 following infection is shown. Images shown are 2 to 3 representative mice from 3 to

6 mice/group. The experiment shown is one of two IL-lp, four IL-18, or four IL-18R independent

experiments. (B) P values (t test) comparing luciferase activity for each knockout strain to C57BL/6J are

<0.05. (C) Aggregate survival curve of IL-JR (n = 9)-, IL-18 (n = 19)-, and IL-18R (n = 13)-knockoutmice compared with WT C57BL/6J control mice (n = 13). (D) IL-18 measurements in serum of deficient

mice on day 5 after infection. No detectable levels of IL-ID were detected in circulation.

59

Discussion

Generation of a robust innate immune response is required to orchestrate murine

resistance against the intracellular pathogen Toxoplasma gondii, as well as a wide spectrum of

other pathogenic agents (Hunter and Sibley 2012). Resistance to Toxoplasma infection is

critically dependent on the TLR-associated adaptor protein MyD88 and induction of IL- 12, IFN-

y, and the synthesis of nitric oxide (NO). In this study, we show that in vivo generation of host

protective immunity against Toxoplasma is also highly dependent on the inflammasome sensors

NLRP 1 and NLRP3 and the secretion of the caspase- 1-dependent proinflammatory cytokine IL-

18. Infection of mice deficient in NLRP3, NLRPlabc, caspase-1/11, or the inflammasome

adaptor protein ASC led to decreased levels of circulating IL-18, increased parasite replication,

and death. Using mice deficient in IL-18 and IL-18R, we show that this cytokine plays an

important role in limiting parasite replication to promote murine survival.

IL- 18, like IL- 10, has been extensively linked to both protective immune responses and

disease induction. IL- 18 mediates enhancement of innate resistance to acute toxoplasmosis by

triggering IFN-y induction in immune cells, especially T and NK cells, and works in synergy

with IL-12. It has previously been used as a protective treatment (Cai, Kastelein, and Hunter

2000; Yap et al. 2001), and IL-18 depletion by antibodies significantly alters murine

susceptibility upon infection with lethal doses of Toxoplasma (Mordue et al. 2001). In fact, IL- 18

was at one time known as "IFN-y-inducing factor" (Okamura et al. 1995). The inactive pro-IL- 18

form is constitutively expressed in a wide range of cell types (Puren, Fantuzzi, and Dinarello

1999) but requires processing by caspase-1 to promote its secretion, as evidenced by the

drastically depleted levels of IL-18 during infection of caspase-1/11 -/- mice (Figure 2D), and

promote the induction of IFN-y production in vivo (Ghayur et al. 1997; Gu 1997).

60

Interestingly, Asc-deficient mice produced even less circulating IL-18, indicating that other

factors such as caspase-8 (Bossaller et al. 2012) may contribute to IL-i 8 processing.

The role of IFN-y in resistance to Toxoplasma is extensively documented (Hunter and

Sibley 2012; Melo, Jensen, and Saeij 2011). IFN-y activates cellular pathways that promote

resistance to toxoplasmosis through multiple mechanisms, including activation of the interferon-

inducible GTPases (IRG-GTPases) (Howard, Hunn, and Steinfeldt 2011; Hunn et al. 2011) and

NO regulation (Scharton-Kersten 1997), processes certainly dependent on induction of IFN-y by

IL-18 produced following NLR inflammasome activation. However, IL-18 has also been linked

to pathology during infection with type I strains of Toxoplasma, and IL-18 depletion resulted in

enhanced survival by limiting the propathologic immune response that these virulent strains

induce (Mordue et al. 2001; Gavrilescu and Denkers 2001). Hence, the balance between the

protective and pathological roles of IL-18 is likely highly dependent on mouse genetics,

Toxoplasma strain differences, challenge doses, routes of infection, and rates of disease

progression. Previous work using a low-dose type II (PTG) infection in caspase-l-deficient mice

(now known to be caspase-1/11 deficient) concluded that these mice were not altered

in Toxoplasma susceptibility relative to wild-type control mice (Hitziger et al. 2005). However,

this study was performed with a parasite dose that does not typically induce measurable levels of

systemic IL-18 (Mordue et al. 2001), and the mice used had a mixed 129/B6 background, which

itself may influence pathogenesis. Our results, using mice sufficiently backcrossed onto a

C57BL/6NTac background and a parasite inoculum that induces systemic IL- 18, show a role for

caspase- 1 and IL- 18 in murine resistance. IL- 18 concentrations are known to vary through the

course of infection and are clearly dependent on the parasite strain and inoculum used (Mordue

et al. 2001; Gavrilescu and Denkers 2001). Our results suggest that this cytokine plays a pivotal

61

role in mediating acute toxoplasmosis, with the cytokine playing an important early role in the

control of parasite replication (Figure 5B). How exactly IL- 18 mediates this protection requires

further studies. Later in infection, however, high levels of IL-18 have previously been shown to

cause dysregulated induction of propathologic cytokine levels that contribute to lethality in high-

dose, virulent infections (Mordue et al. 2001; Gavrilescu and Denkers 2001).

The recruitment of inflammatory monocytes to sites of infection is essential to control

parasite growth and dissemination in murine models of toxoplasmosis (Dunay et al. 2008;

Robben et al. 2005). In rats, control of parasite proliferation and dissemination in vivo is

controlled by the Toxol locus (Cavailles et al. 2006). Our recent work showed that macrophages

from Toxoplasma-resistant rat strains (e.g., LEW and SHR) undergo pyroptosis in response to

inflammasome activation induced by parasite infection, and this rapid cell death is sufficient to

limit parasite replication and promote sterile cure (Cirelli et al. 2014). In previous work by Miao

et al., caspase-1-induced pyroptotic cell death was also identified as an innate immune

mechanism to protect against intracellular pathogen infection (Miao et al. 2010). In their study,

the authors used a panel of mouse strains deficient in IL-i p, IL-18, IL-I PR, or various

combinations of those to show a dispensable role for IL-ip and IL-18 in the clearance

of Salmonella enterica serovar Typhimurium that expresses flagellin, suggesting that innate

control of bacterial infection is occurring by pyroptosis, without a requirement to induce an overt

inflammatory response. In this study, we show that in vitro Toxoplasma infection of murine bone

marrow-derived macrophages primarily activates the NLRP3 inflammasome, resulting in the

rapid production and cleavage of IL-ip, but does not induce pyroptosis. Interestingly,

although Toxoplasma-infected macrophages showed efficient caspase- 1/11-dependent IL-ip

cleavage and secretion, these cells did not upregulate, cleave, or secrete their preexisting pools of

62

IL-18. Furthermore, splenocytes also did not show any IL-18 cleavage following infection.

Paradoxically, significant concentrations of IL-ip were not detected following infection in any

mouse strain, whereas high levels of IL-18 were found in serum in all infection studies that we

performed with different Toxoplasma strains. Because activation of the murine inflammasome

does not affect Toxoplasma growth in macrophages (data not shown) and does not induce

pyroptosis, our results suggest that IL-18 activation and not pyroptosis is the genetic basis for in

vivo inflammasome-mediated control of parasite proliferation. Although the in vivo cellular

source for this inflammasome-generated IL-18 is not known, it is likely of nonmyeloid origin,

and bone marrow chimera studies should be performed to address this question. Our results also

suggest that NLRP 1-mediated events may be more important in vivo and that activation of

NLRPI may likewise occur in cells other than macrophages.

The role of inflammasome activation in the pathogenesis of Toxoplasma infection in

human infection has recently been suggested (Jamieson et al. 2010; Witola et al. 2011).

Polymorphisms in the human NLRP1 gene are associated with susceptibility to congenital

toxoplasmosis, and NLRP1 contributes to controlling parasite growth in human monocytes. A

recent study in human macrophages provides compelling evidence that the inflammasome

components ASC and caspase-1 regulate the release of IL-is and that the type II allele of the

parasite dense granule protein GRA15, which activates NF-KB nuclear translocation, is necessary

for maximal induction of this cytokine (Gov et al. 2013). Indeed, our in vitro infection data show

that Toxoplasma murine inflammasome-mediated secretion of IL-i IP is strain dependent and that

only parasites expressing the GRA15 type II allele, which directly activates NF-KB, were able to

induce secretion of IL-ip in unprimed BMDMs (Figure 1). While the relative and contributing

roles of IL- IP compared to IL-18 remain to be determined in the control of acute toxoplasmosis,

63

our preliminary studies using IL-I receptor-knockout mice on a mixed background argue that IL-

1p plays a less significant role in the control of parasite infection than it does in IL- 18 or IL- I8R

knockouts (Figure 5). In vivo administration of IL-ip in LPS-primed caspase-1/1 1-deficient

mice has previously been shown to increase IL-6 (Gu 1997), so it is conceivable that IL-IP

functions locally to induce increased levels of IL-6 capable of altering inflammation-induced

changes in myeloid output that impact Toxoplasma pathogenesis (Chou et al. 2012).

Of the two NLR inflammasomes activated, we found that murine resistance to acute

infection was principally dependent on activation of the NLRP3 receptor both in

vitro (Figure 1J) and in vivo (Figure 3). Several reports have linked P2X(7) receptor, a potent

activator of the NLRP3 inflammasome, with control of acute toxoplasmosis (Jamieson et al.

2010; Lees et al. 2010; Correa et al. 2010; Miller et al. 2011). How and in what cell

type Toxoplasma activates the murine NLRP3 inflammasome or why its activation does not lead

to rapid macrophage death or IL-18 processing is enigmatic. Regulators of the NLRP3

inflammasome include ATP, the guanylate-binding protein 5 (GBP5), cellular stresses that alter

calcium and potassium concentrations, redox status, and the unfolded protein response (UPR)

(Wen, Miao, and Ting 2013). Importantly, Toxoplasma encodes a variety of virulence effector

proteins that specifically inactivate the host endoplasmic reticulum (ER)-bound transcription

factor ATF6p and induction of the UPR during ER stress (Yamamoto et al. 2011), affect the

recruitment of 65-kDa guanylate-binding proteins (GBPs) (Niedelman et al. 2013; Selleck et al.

2013), or alter calcium and potassium efflux to signal Toxoplasma egress (Fruth and

Arrizabalaga 2007), perhaps indicating that the parasite has specifically evolved effector proteins

to minimize NLR inflammasome activation to alter its pathogenesis.

64

Our work also identified Toxoplasma as the second pathogen, after B. anthracis, whose

pathogenesis is altered by expression of the murine NLRPI inflammasome (Boyden and Dietrich

2006). We show that the Nlrpi locus is capable of regulating parasite proliferation in vivo,

with Nlrpi knockout mice possessing significantly higher parasite burdens

following Toxoplasma infection. Although the majority of NLRP1-deficient mice died acutely

(Figure 4), they were, however, less susceptible to infection than were caspase-

1/11, Asc, Nlrp3, IL-18-, or IL-18R-deficient mice in the same genetic background and possessed

only 5- to 10-fold-higher parasite loads than in WT infections. How Toxoplasma activates the

NLRP1 inflammasome is unclear. Activation of rodent NLRP1 inflammasomes by

the B. anthracis lethal toxin (LT) occurs via proteolytic cleavage at a specific consensus

sequence in the polymorphic N terminus of NLRP1 (Levinsohn et al. 2012; Newman et al.

2010). In human infection, NLRP1 polymorphism variants are likewise known to alter the

susceptibility to congenital toxoplasmosis (Witola et al. 2011). One logical hypothesis is that

the Toxoplasma-encoded effector molecule responsible for activation of NLRP1 is, like LT, a

protease Toxoplasma secretes a wide range of proteases (Binder and Kim 2004; Choi, Nam, and

Youn 1989; Dou and Carruthers 2011; Dou, Coppens, and Carruthers 2013; Kim 2004; Shea et

al. 2007), and similar induction of IL-1f observed upon infection of primed BMDMs with

any Toxoplasma strain suggests that the putative protease, or factor responsible for activation of

NLRP1, is not likely to be Toxoplasma strain specific or is at least conserved among the majority

of strains. Alternatively, polymorphisms in Nlrpl could affect the interaction with a different

host "sensor" protein that serves as the adaptor for assembly and activation of the NLRP1

inflammasome, as has been previously described for the NLRC4/NAIP5/NAIP6 inflammasome

recognition of flagellin (Kofoed and Vance 2011; Zhao et al. 2011).

65

In summary, we establish that both NLRP3 and NLRP1 are important in vivo regulators

of Toxoplasma proliferation and that IL- 18 signaling is required to mediate host resistance to

acute toxoplasmosis. Our findings also indicate that innate resistance to acute toxoplasmosis is

dependent on the activation of both TLR and NLR sensors that cooperate to detect the presence

of pathogen products or the danger signals that they induce during active infection. The

identification of the Toxoplasma factor that mediates NLR inflammasome activation may

contribute new insight into the development of therapeutic options to combat this important

human pathogen.

66

Materials and Methods

Ethics statement

All animal experiments were performed in strict accordance with guidelines from the

NIH and the Animal Welfare Act, under protocols approved by the Animal Care and Use

Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of

Health (protocols LPD-8E and LPD-22E), and the MIT Committee on Animal 441 Care

(assurance number A-3125-01).

Material

Ultrapure LPS was purchased from Calbiochem/EMD Biosciences (San Diego, CA).

Luciferin was purchased from Caliper Life Sciences (Hopkinton, MA). Nigericin was purchased

from Invivogen (San Diego, CA). Mycalolide B was purchased from Wako (Richmond, VA).

Mice and NLRP1 expression status-based nomenclature

IL-18- and IL-18 receptor (IL-18R)-knockout mice on the C57BL/6J background (>10

backcrosses) and IL-IR-deficient mice on a partially backcrossed 129 x C57BL/6J background

were obtained from Jackson Laboratories (Bar Harbor, ME). Caspase-1-knockout mice have

been previously described (Sutterwala et al. 2006) and were backcrossed to C57BL/6NTac mice

for 10 generations. These caspase- 1 -knockout mice are also deficient in caspase- 11 (Kayagaki et

al. 2011). Mice deleted for all three Nlrpi genes in the murine Nlrplabc locus (C57BL/NTac

background), as well as those deleted only for Nlrpib (C57BL/6J background), have been

previously described (Kovarova et al. 2012; Masters et al. 2012). Mice deleted at

67

Nlrp3 (C57BL/6J background) (Mariathasan et al. 2006) and Asc (C57BL/6J background)

(Sutterwala et al. 2006) have been previously described.

Parasites

Tachyzoites from luciferase-expressing type I (RH) and type II (76K or

Prugniaud) T. gondii parasites were used for all studies. The following strains (haplogroup/type

in parentheses) were used in a survey of effects on murine BMDMs: GT1 (I), ME49 (II), DEG

(II), CEP (III), VEG (III), CASTELLS (IV), MAS (IV), GUY-KOE (V), GUY-MAT (V), RUB

(V), BOF (VI), GPHT (VI), CAST (VII), TgCATBr5 (VIII), P89 (IX), GUY-DOS (X), VAND

(X), Cougar (XI), B41 (XII), B73 (XII), RAY (XII), and WTD3 (XII). The generation of

luciferase-expressing parasites using the plasmid pDHFR-Luc-GFP gene cassette has been

described previously (Saeij et al. 2005). To construct the RH, Prugniaud, and 76K GFP-LUC

strains, pDHFR-Luc-GFP was linearized with NotI, parasites were electroporated, and those with

stable GFP expression were isolated by fluorescence-activated cell sorting and cloned by limiting

dilution. Generation of Pru GRA15-knockout (KO) parasites has been previously described

(Rosowski et al. 2011). All parasite strains were routinely passaged in vitro in monolayers of

human foreskin fibroblasts (HFFs) at 37'C in the presence of 5% CO 2 and quantified by

hemocytometer counts prior to infection studies. In some experiments, mycalolide B (3 pM,

20 min) was used to pretreat isolated parasites prior to washing in phosphate-buffered saline

(PBS) (3x) before infections.

68

Cell culture

Bone marrow-derived macrophages (BMDMs) were cultured in complete Dulbecco's

modified Eagle's medium (DMEM) with 20% L929 cell culture supernatant for 7 days. L929

mouse fibroblast cells were grown in DMEM supplemented with 10% fetal bovine serum,

10 mM HEPES, and 50 pg/ml gentamicin (all obtained from Invitrogen, Carlsbad, CA) at 37*C

in 5% CO 2. BMDMs with or without LPS priming (0.1 ig/ml, 2 h) were infected

with Toxoplasma at various multiplicities of infection (MOIs), and cell viability was assessed at

24 h using the CellTiter 96 AQueous One Solution cell proliferation assay (Promega, Madison,

WI). Culture supernatants were removed for cytokine measurements by enzyme-linked

immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN) or Western blotting,

following concentration using Amicon filters (3,000-molecular-weight cutoff) (Millipore,

Billerica, MA) or Spin-X UF 500 concentrators (5,000-molecular-weight cutoff) (Coming,

United Kingdom). For Western blots, anti-mouse IL-lp (Abcam, Cambridge, MA) or anti-

caspase-1 antibody (Abcam, Cambridge, MA) was used as the primary antibody. Secondary

antibodies were from Jackson Immunoresearch (West Grove, PA). Immun-Star Western C

substrate (Bio-Rad, Hercules, CA) and a charge-coupled device camera (Chemidoc XRS; Bio-

Rad) were used for visualization. All immortalized macrophage cell lines (WT and caspase-

1/1 1'-) were grown in complete DMEM with 10% L929-conditioned medium.

Microarray analysis

Microarray analyses were performed as previously described (Jensen et al. 2011).

69

Mouse infections

Mice (male and female, 8 to 12 weeks old) were infected intraperitoneally (i.p.) with either

10,000 (76K) or 1,200 (Pru) type II tachyzoites diluted in 400 pl of phosphate-buffered saline.

Mice were imaged on successive days (typically days 4 to 9) postinfection, and parasite burden

was quantified by firefly luciferase activity using an IVIS BLI system from Caliper Life

Sciences. Mice were injected i.p. with 3 mg of D-luciferin substrate (prepared in 200 pl of PBS)

and imaged for 5 min to detect photons emitted, as previously described (Saeij et al. 2005). Mice

were bled by tail vein at day 5 and/or day 9 after infection. Blood collection was performed in

either serum collector or Microtainer EDTA tubes (Sarstedt, Newton, NC). IL-10 and IL- 18 were

measured by ELISA (R&D Systems, Minneapolis, MN).

70

Supplemental Figures

A1000

* -LPS800 +LPS600

- 400

200

T T

0 6 10 24Time (Hours)

B120 -LPS

-C 100 + LPS75

800)6020

0 6 10 24ime (Hours)

Supplementary Figure SI. Type II parasites activate the inflammasome without inducing cell death.

BMDMs were primed with 100 ng/ml LPS or left unstimulated for 2 hours and subsequently infectedwith type II parasites (Pru; MOI, 0.4) for 24 h. (A) Quantification of IL-I 1 in supernatants was performed

using ELISA. (B) Cell viability of infected cells in panel A was determined at different time points usingan MTS [3-(4,5-dinmethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Hl-tetrazoliumn]assay.

71

______ ____________________________ __

Type I Type IVLPS LPS

37kD

17kD

Supplementary Figure S2. Type I and type IV parasites induce cleavage of pro-IL-p. C57BL/6BMDMs were primed with 100 ng/ml LPS for 2 hours and infected with type I (RH) or type IV (MAS)parasites (MOI, 5) for 24 hours. Western blot analysis was performed on concentrated supernatant (25-fold), probing for pro-IL-lp (37 kDa) and active IL-1 (17 kDa).

72

200

E150CD

'-100ca

- 50

T

-I-

0N

04Z0annn m

Supplementary Figure S3. Type II parasites activate the Nlrp3 inflammasome. IL- I3 secretion from

BMDMs prepared from wild-type C57BL/6 mice (blue) or C57BL/6 mice lacking Nlrplb (red), Nlrp3

(green), or Nlrpl b and Nlrp3 (orange) primed for 4 h and then infected with type I parasites (Pru; MOI,

0.8) for 24 hours. The figure represents one experiment. Error bars, + standard deviations.

73

Q

Acknowledgments

This work was supported in part by the Intramural Research Program of the NIH and

NIAID (M.E.G., S.H.L., and A.S.). G.G. was supported by a research fellowship award from the

Crohn's & Colitis Foundation of America (CCFA) and is a CCFA Helmsley Scholar. K.M.C.

was supported by NIH grant A1l04170. J.P.J.S. was supported by RO1-AI080621 and a Pew

Scholar in the Biomedical Sciences Award. M.E.G. is a scholar of the Canadian Institute for

Advanced Research (CIFAR) Program for Integrated Microbial Biodiversity.

74

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Chapter Three:Inflammasome sensor NLRP1 controls rat macrophage

susceptibility to Toxoplasma gondii

Kimberly M. Cirelli, Gezahegn Gorfu, Musa A. Hassan, Morton Printz, Devorah Crown,

Stephen H. Leppla, Michael E. Grigg, Jeroen P.J. Saeij, Mahtab Moayeri

Address correspondence to Michael E. Grigg, griggm@niaid.nih.gov; Jeroen P.J. Saeij,

j sacij@ucdavis.edu; or Mahtab Moayeri, mmoayeri@niaid.nih.gov.

Kimberly M. Cirelli contributed to experiments in Figure lB and D, 2, 3, 4A, C-G, 5B, E, F, 6E-

G, S1, S2, S3, S4, S5 and S6. Gezahegn Gorfu contributed to experiments in Figure 4B. Musa A.

Hassan contributed to experiments in Figure 2, S3 and S4. Mahtab Moayeri contributed to

experiments in Figure lB-E, 2, 5A-D, F, 6A-D, H and I, S7 and S8.

Originally published in PLoS Pathogens 10(3). March 13, 2014

Supplementary Data is available online at

http://ioumals.olos.org/plospathogens/article?id=10.1371/ioumal.ppat.1003927#s5

82

Abstract

Toxoplasma gondii is an intracellular parasite that infects a wide range of warm-blooded

species. Rats vary in their susceptibility to this parasite. The Toxol locus conferring Toxoplasma

resistance in rats was previously mapped to a region of chromosome 10 containing Nlrpi. This

gene encodes an inflammasome sensor controlling macrophage sensitivity to anthrax lethal toxin

(LT) induced rapid cell death (pyroptosis). We show here that rat strain differences in

Toxoplasma-infected macrophage sensitivity to pyroptosis, IL-1p/IL-18 processing, and

inhibition of parasite proliferation are perfectly correlated with NLRP1 sequence, while inversely

correlated with sensitivity to anthrax LT-induced cell death. Using recombinant inbred rats, SNP

analyses and whole transcriptome gene expression studies, we narrowed the candidate genes for

control of Toxoplasma-mediated rat macrophage pyroptosis to four genes, one of which was

Nlrpl. Knockdown of NlrpJ in pyroptosis-sensitive macrophages resulted in higher parasite

replication and protection from cell death. Reciprocally, overexpression of the NLRP1 variant

from Toxoplasma-sensitive macrophages in pyroptosis-resistant cells led to sensitization of these

resistant macrophages. Our findings reveal Toxoplasma as a novel activator of the NLRPI

inflammasome in rat macrophages.

83

Author Summary

Inflammasomes are multiprotein complexes that are a major component of the innate

immune system. They contain "sensor" proteins that are responsible for detecting various

microbial and environmental danger signals and function by activating caspase- 1, an enzyme that

mediates cleavage and release of the pro-inflammatory cytokines, IL-10 and IL- 18. Toxoplasma

gondii is a highly successful protozoan parasite capable of infecting a wide range of host species

that have variable levels of resistance. Rat strains have been previously shown to vary in their

susceptibility to this parasite. We report here that rat macrophages from different inbred strains

also vary in sensitivity to Toxoplasma-induced lysis. We find that NLRP1, an inflammasome

sensor, whose only known agonist is anthrax LT, is also activated by Toxoplasma infection. In

rats there is a perfect correlation between NLRP1 sequence and macrophage sensitivity to

Toxoplasma-induced rapid cell death, inhibition of parasite proliferation, and IL-1p/IL-18

processing. Nlrpl genes from sensitive rat macrophages can confer sensitivity to this rapid cell

death when expressed in Toxoplasma resistant rat macrophages. Our findings suggest

Toxoplasma is a new activator of the NLRP1 inflammasome.

84

Introduction

Toxoplasma gondil is an obligate intracellular parasite, for which different host species or

strains within a species display variable susceptibilities. Different Toxoplasma strains also differ

in virulence within the same host, suggesting variation in effectors among parasite strains and/or

their impact in various hosts. Host innate immunity is known to play a critical role in

susceptibility to infection. In mice, for example, resistance to Toxoplasma infection is critically

dependent on the induction of IL-12, which subsequently induces IFN-y, the main mediator of

toxoplasmicidal activities (Melo, Jensen, and Saeij 2011).

Rats, like humans, are quite resistant to Toxoplasma infection when compared to mice.

However varying levels of resistance also exist among rat strains. The resistance of the Lewis

(LEW) strain is characterized by total clearance of the parasite, failure to develop cysts and the

absence of a strong antibody response. Fischer (CDF) and Brown Norway (BN) rats, however,

are susceptible to chronic infection and develop transmissible cysts in their brain and muscle

tissue (Cavailles et al. 2006; Sergent et al. 2005). Resistance in rats is a dominant trait and is

linked to myeloid cell control of parasite proliferation (Cavailles et al. 2006; Sergent et al. 2005).

Linkage analyses of LEWxBN F2 progeny was previously used to

map Toxoplasma resistance in rats to a single genetic locus, termed Toxol, within a 1.7-cM

region of chromosome 10 (Cavailles et al. 2006). We noted that this locus overlaps with the

locus that controls rat and macrophage sensitivity to the anthrax lethal toxin (LT) protease.

Inbred rat strains and their macrophages exhibit a perfectly dichotomous phenotype in response

to LT: animals either die rapidly (<1 hour) or exhibit complete resistance to the toxin (Newman

et al. 2010). Only macrophages from LT-sensitive rat strains undergo rapid caspase-1 dependent

death (pyroptosis). The HXB/BXH recombinant inbred (RI) rat collection, developed from the

85

, . b, Lb.,V- -

SHR/Ola and BN-Lx congenic parental strains (Pravenec et al. 1996; Pravenec et al. 1989; Printz

et al. 2003), with opposing LT sensitivities, was used to map anthrax toxin susceptibility to a

single locus at 55.8-58.1 Mb of rat chromosome 10. SNP analyses and sequence correlation to

phenotype implicated the inflammasome sensor Nirpi (nucleotide-binding oligomerization

domain, leucine-rich repeat protein 1) as the likely susceptibility locus. NLRP1 is a member of

the NLR cytosolic family of pathogen-associated molecular pattern molecule (PAMP) sensors,

the activation of which leads to recruitment and autoproteolytic activation of caspase-1, followed

by cleavage and release of the proinflammatory cytokines IL-1p and IL-18. NLR-mediated

activation of caspase-1 is typically accompanied by rapid death of macrophages through a

process known as pyroptosis (Lamkanfi and Dixit 2012; Song and Lee 2012). NLRP1 sequences

from 12 inbred rat strains show a perfect correlation between sensitivity and the presence of an

N-terminal eight amino acid (aa) LT cleavage site (Newman et al. 2010; Levinsohn et al. 2012).

Proteolytic cleavage by LT activates the NLRP1 inflammasome in rat macrophages leading to

rapid caspase-1 dependent cell death (pyroptosis) and cytokine processing (Levinsohn et al.

2012).

We hypothesized that the Toxol locus could be Nlrpi as the macrophage is an important

carrier of the parasite (Mordue and Sibley 2003; Suzuki et al. 2005) and inflammasome-mediated

pyroptosis of this cell could impact in vivo parasite dissemination. The recent association of

polymorphisms in the human NLRPJ gene with susceptibility to congenital toxoplasmosis,

evidence that P2X(7) receptors influence parasite proliferation in mouse cells, and the finding

that IL-i I3 responses in Toxoplasma infected human monocytes are dependent on caspase- 1 and

the inflammasome adaptor protein ASC all suggest that the inflammasome plays a role in

86

determining the outcome of Toxoplasma infection in humans and mice (Lees et al. 2010; Witola

et al. 2011; Gov et al. 2013).

Our results indicate that rat strain macrophages exhibit dichotomous susceptibilities to

Toxoplasma-induced rapid lysis and associated cytokine processing in a manner correlated with

NLRP1 sequence. We go on to show that NlrpJ knockdown in Toxoplasma-sensitive

macrophages protects against this cell death while overexpression of certain variants of the gene

in resistant macrophages can sensitize these cells to the parasite-induced pyroptosis. Our findings

establish Toxoplasma as the second known activator of the inflammasome sensor NLRP1 and

suggest a mechanism of host resistance involving activation of this sensor.

87

Results

NLRP1 sequence in inbred rats correlates with macrophage cell death, parasite

proliferation and IL-1PIL-18 release

The Toxol locus on chromosome 10, which controls rat resistance to toxoplasmosis, maps

within a region containing the inflammasome sensor Nlrpi gene. NLRP1 was previously shown

to control rat macrophage sensitivity to pyroptosis by the anthrax protease LT. Sequencing of

twelve inbred rat strains revealed five highly homologous variants, two encoding NLRP 1 protein

sensitive to LT-mediated cleavage activation (NLRPl varianti,2), and three which encode LT-

resistant proteins (NLRPlvarian t3,4 ,5 ) (Figure 1A). We noted that rat strains encoding

NLRPvariantl, 2 historically support parasite proliferation in myeloid cells while rat strains

encoding NLRP lvariant do not (Cavailles et al. 2006). Therefore we investigated whether

macrophages from rats expressing different NLRP1 variants also differed in inflammasome

activation and pyroptosis upon parasite infection. Inflammasome activation was assessed by

monitoring cell death and cleavage of pro-IL-1p (37 kD) with subsequent secretion of mature

active IL-i IP (17 kD). We infected BMDMs from LT-sensitive CDF, BN or SD (NLRP lvariantl,2)

rat strains and LT-resistant LEW and SHR (NLRPlvariant5) rat strains with luciferase-expressing

Type I (RH) and Type 11 (76K, or PRU) Toxoplasma strains at various MOIs. BMDM viability

measurements showed that NLRP1 vaants-expressing macrophages underwent a rapid cell death

after Toxoplasma infection starting at 3 hours and completed by 24 hours whereas the majority

of the NLRP 1variantl,2 -expressing macrophages remained viable and

supported Toxoplasma growth even 24 hours after infection (Figure 1B-D). The parasite itself

did not contribute significantly to MTT or LDH signals (Figure S1, panels A, B) and DAMPs

88

from lysed host cells also did not induce cell death (Figure Si panels C, D). Results were

unaltered when cells were pre-treated with LPS (100 ng/ml) prior and throughout infection

(Figure SI panels C, D). Fischer F344/NTac (NLRPlvaIan) macrophages also showed

resistance similar to that of Fischer CDF macrophages (data not shown). Both NLRPlvariant1, 2 and

NLRP Ivariant -expressing macrophages were fully responsive to nigericin-induced NLRP3

activation (Figure S2 and (Newman et al. 2010)), indicating fully functional inflammasome

assembly and caspase- 1 function in these rat strains.

89

UNLRR CARD S D

CDFF344

EWSHR

~LEW~SHR*CDP

BN

C

100

50

030

1

Macrophage pyroptosis

LT T. gondii

S R

S R

~.

76K

0 10 20Time post infection (hrs)

E*LEW

- C

PRU 40 10 20

Time post infection (hrs)

100,

50.

30 0

* HXBI+ HXB29-. HXB15

76K

10 20Time post infection (hrs)

Figure 1. NLRPI sequence in inbred and RI rats correlates with rapid macrophage death.(A) Sequence map of rat NLRPI variants. This diagram was modified from (Newman et al. 2010).Vertical black lines indicate amino acid polymorphisms relative to the protein encoded by allele I.Approximate NACHT, LRR and CARD domain locations relative to polymorphisms are shown.Macrophage sensitivity to LT-induced pyroptosis for the listed rat strains is from (Newman et al. 2010)and Toxoplasma sensitivities are from this work. (B-E) Viability measurements for rat BMDMs from

LEW, SHR (expressing NLRP vaa.1 5

); CDF, BN or SD (expressing NLRPlvarant1 2 ); or RI rat strainsfollowing infection with Toxoplasma Type I (RH) or Type II (76K or PRU) (MOI, 3) by MTT

measurements. Data shown are average from three independent experiments with SD (triplicate

wells/experiment/condition), except RI strains, which are averages from two experiments (triplicatewells/experiment/condition). Viability values were calculated relative to MTT measurements foruninfected control cells at each time point, which were set at 100%. P-values comparing all

NLRPI va1an -expressing strains to NLRP I ,,-"t -expressing strains are <0.00 1.

90

A

1

2!'

s Ill

NACHT

11 I 111

B

g50.

R S

+ LEWSHR

* CDFON

RH

0 10 20Time post infection (hrs)

D

5

30

1004

50-

030

100

We next tested macrophages from three rat strains (HXB 1, HXB15 and HXB29) from the

HXB/BXH recombinant inbred (RI) rat collection previously used to map LT

sensitivity (Newman et al. 2010). These strains have chromosome 10 crossover points closely

flanking the Nlrpi locus, as indicated by SNP analyses. We found that macrophages from the RI

strain HXB 1, an LT-resistant strain, were sensitive to Toxoplasma Type I (RH) and Type II

(76K) infection-induced lysis while the macrophages from the other two strains, which are LT-

sensitive, were resistant to parasite induced rapid death (Figure lE). These rats allowed us to

reduce the Toxol locus from the previous 54.2 Mbp-61.8 Mbp region to 54.2 Mbp-59.2 Mbp

(Figure S3). We performed SNP and haplotype analyses for the CDF (F344/Crl), F344/NTac,

BN (all strains with macrophages resistant to Toxoplasma-induced lysis) and the SHR strain (a

strain with macrophages sensitive to Toxoplasma-induced lysis) and further narrowed the region

determining resistance to 55.3-59.2 Mbp (between SNPs rs63997836 and rs106638778) (Figure

S3). This region contained 133 genes of which 21 contained non-synonymous SNPs that were

present in F334 and/or SHR rats, where genotype correlated with Toxoplasma resistance

phenotype. To further narrow down the list of possible candidate genes, we performed whole

transcriptome sequencing on BMDM from the LEW (pyroptosis-sensitive macrophages), BN

(pyroptosis-resistant macrophages) and SD (pyroptosis-resistant macrophages) strains. We

determined which genes were expressed in unstimulated and LPS-stimulated LEW BMDM

(which are sensitive to parasite induced pyroptosis under both conditions), and contain SNPs that

correlate with the resistance phenotype. Sixty-five of the 133 genes in the fine-mapped region

were expressed (fragments per kilobase of transcript per million mapped reads >2) but only five

of these contained non-synonymous SNPs that distinguished LEW from SD/BN (Dataset

S1 and Figure S4). Although there were also differences in gene expression levels between

91

LEW and SD/BN macrophages, none of the genes were expressed higher (1.5 fold) in both the

non-stimulated and LPS stimulated LEW macrophages compared to the SD/BN macrophages

(Dataset Si). By combining all analyses, we were able to narrow down the possible candidate

genes to Aurkb (Aurora kinase B1, 55.7 Mbp, 1 SNP), Neurl4 (neutralized homolog 4, 56.7 Mbp,

1 SNP), Cxcli6 (chemokine C-X-C ligand 16, 57.3 Mbp, 1 SNP) and Nlrpi (6 SNPs). Figure 2

summarizes the above described mapping steps. Of these four genes, Nlrpi was the most likely

candidate to be Toxo; it contained the highest number of non-synonymous SNPs and is a known

activator of the inflammasome. Our fine-mapping analyses combined with the established perfect

correlation between sensitivity to Toxoplasma induced macrophage cell death and the NLRP1 N-

terminal sequence in inbred and RI rats (Newman et al. 2010), which was in turn inversely

correlated to rat resistance to chronic, transmissible Toxoplasma infection suggested that the

Toxol locus could be the NIrpI gene.

92

Original Toxol locus

uti 133 genes a6LA LA

LA21 genes L

!0 +PL A r.0

4 candidates

000

CD

RI and Inbred SNPs

F344 vs. SHRNonsynonymous SNPs

Whole transcriptomesequencing

Figure 2. Summary flow diagram for mapping of rat macrophage sensitivity to four candidategenes. Methods for reducing the number of candidates at each stage are listed to the right and explainedin detail in the Results section. Detailed SNPs and gene lists for each stage can be found inSupporting Figures S3, S4 and Dataset SI.

IA survey of Toxoplasma strains that are genetically distinct from the archetypal 1, I and III

strains (Minot et al. 2012; Su et al. 2012) showed that they all induced NLRP I variant-dependent

rapid cell death (Figure 3).

93

Cc-4

0 n

0U)I'0

Ia-J

1.0.

0.8.

0.6

0.41

0.2.

0.0

"'LEW* SD

*n I

gi li' *ai e il Li LI

CO'4

Figure 3. NLRPI variant-dependent rapid cell death is induced by many different parasite strains.Viability as measured by LDH release for BMDMs from SD (NLRP1 2 variant) or LEW (NLRP1 5 variant)infected with strains representing global diversity for 24 hours (MOI, 0.5-1 depending on strain, n = 4wells/strain). P-values comparing LEW and SD <0.05 for all strains except MAS, CAST, GPHT andGUY-MAT.

Because cell death was consistently dependent on MOI, we tested whether parasite invasion

was required for cell death, as Toxoplasma can secrete effectors from its rhoptry organelles

directly into the host cytoplasm. Parasites treated with Mycalolide B, a drug that blocks invasion

but allows for secretion of microneme and rhoptry contents, attached but were unable to kill

BMDMs, indicating that macrophage sensitivity to cell death was invasion-dependent (Figure

4A). Mycalolide B did not affect the viability of parasites or their ability to secrete rhoptry

contents as verified by the observation that every cell with an attached mycalolide-B-treated

parasite also had protein kinase ROP16 activation of STAT6 (Figure S5).

Because Toxoplasma needs host cells for replication and the parasite replicates equally well in

fibroblasts from different rat strains (Cavailles et al. 2006), we hypothesized that rapid

macrophage cell death prevents loxoplasma replication. We therefore investigated parasite

94

A

N A - RIRV- " 04j;14

0IRA C2

proliferation in BMDMs from the different rat strains. Toxoplasma burden, as measured by

bioluminescence, was significantly higher in infected NLRPlvariant], 2 -expressing BMDMs than

NLRPI varan -expressing cells (Figure 4B, 4C). This difference was independent

of Toxoplasma strain but perfectly correlated with NLRP1 sequence and continued to increase

over time only in the cell death-resistant BMDMs from Toxoplasma susceptible rat strains

(Figure 4C). Similarly, GFP signal indicative of parasite load was higher in resistant cells from

these rat strains (data not shown). Parasite proliferation was independent of LPS-priming (data

not shown) and more parasites/vacuole were detected in NLRP 1vaan 1,2 -expressing macrophages

compared to NlrpIvaiant 5 -expressing cells (Figure 4D). Although only ~10% of sensitive LEW

(NLRPvaianO ) BMDMs were intact after 24 hours of infection (Figure 4E left panels), 90% of

these surviving cells contained single parasites (Figure 4E right panels). Nearly 100% of

resistant SD, BN or CDF (NLRPIvaianl,2) BMDMs were intact after 24 hours, and >60% of those

infected contained multiple parasites per vacuole (Figure 4D, 4E). To determine if parasites

released from lysed cells were viable, we measured the parasite's ability to reinvade macrophages

by adding an antibody specific for the Toxoplasma surface protein, SAG 1, to the medium of pre-

infected BMDMs. We found that -35% of intracellular parasites in the sensitive LEW BMDMs

were coated with the SAGI antibody while only 5% were coated in resistant cells, demonstrating

that some fraction of parasites released from rat BMDMs that rapidly lyse remain viable and

capable of re-invasion (Figure S6). We verified that SAGI was not shed upon invasion by

immunofluorescence, where 100% of parasites were stained for SAGI when infected SD

BMDMs were fixed and permeabilized at 18 hours post-infection (Figure S6). Supernatants

from lysed Toxoplasma-sensitive BMDMs also did not contribute to the rapid pyroptosis of

resistant macrophages (Figure 4F) or alter parasite proliferation within these cells (Figure 4G).

95

A100

60.40

20

NT Myca-8

D

B

F~3

2

S0

M

E

C400.5 -- SOfR)

~300 E3W -LEWI S)

i 200

100

0-

Time post infection (hrs}

G100 0 CDF80- 0 BNW0 Q ENoSSD

W0 LEW

40' SHR2 0.

Pr 1s .a q,Parasites/vacuole

100.

50

010CDF CDF(Pru) (Pru)

+Super

$A

C

100 C3 MEDIA80- U UNINFECTED LEW

0 IWECTIED LEW T T

40-

0

Parasites/vacuole

Figure 4. NLRPI-variant dependent macrophage death depends on parasite invasion and controlsparasite proliferation.(A) Viability of LEW BMDMs infected with Mycalolide-treated (3 PM, 15minutes) RH tachyzoites (MOI 1) after 24 hours as measured by MTS assay (P-value comparingMycalolide group to untreated = 0.0002). (B, C) Radiance emission analyses of metabolically active,viable Type 11 Toxoplasma 76K parasites (B, graph MOI 3, 6 hours; inset shows representative plate fromone experiment) or Type I RH parasites (C, MOI I over 48 hours) in BMDMs from different rat strains.P-value comparing NLRPI va'an--expressing strains to NLRP Ivaant-expressing strains are <0.01 in I byt-test and <0.0001 in J by two-way ANOVA. (D) Number of parasites/vacuole in infected BMDMS (24hours, MOI 3) as assessed by microscopy is shown. CDF, BN infections were with 76K, and SD, LEWinfections were with RH. Between 50-100 vacuoles counted per experiment. Average values from 3experiments are shown for all strains, except SD (n = 2). P-values are <0.01 (two-way ANOVA) whencomparing NLRPI varant,-expressing strains to NLRPlvanan t5-expressing strains. (E) Left panels show lightmicroscopy images of CDF and LEW monolayers infected with 76K (MOI 6, 6 hours). Right panels showfluorescence microscopy image of single SD and LEW BMDMs infected with RH (MO! 1, 2 hours). Blueis Hoechst stained nucleus, green are GFP-expressing parasites. Dividing parasites in SD cells (upperright) or a single parasite in LEW cells (lower right) are shown. (F) LEW BMDMs were infected withPRU (MOI 3) and at 5 hours post infection culture supernatants from dying cells was spun, filtered andtransferred to similarly infected (PRU, MOI 3) CDF BMDMs. Viability of CDF BMDMs was assessed at10 hours post-infection by MTT staining. All values were calculated relative to uninfected controlBMDMs (G) SD BMDMs were infected with RH parasites (2 hours, MOI 1), washed with PBS andmedium replaced with fresh media, media from RH-infected (24 hours, MOI 1) or uninfected LEWBMDMs. Parasites/vacuole counted at 24 hours. P-values >0.1 (ns) for comparison of any of three groupsfor 1, 2, 4 and 8 parasites/vacuole counts (by two-way ANOVA).

96

To investigate whether Toxoplasma infection induced maturation and secretion of IL- I P

and IL-18 in an NLRP1 sequence-dependent manner, we measured secreted levels of these

cytokines in the different rat strains. In the absence of LPS priming, Type II strain-infected

BMDMs did not produce IL-1f (data not shown), but low levels of IL-18 were measurable by 6

hours (PRU) and 24 hours (76K) of infection in an NLRP 1 variant-dependent manner. Thus in

the unprimed situation, both 76K and PRU produced a much higher response in the LEW

macrophages (expressing NLRPlvariants) when compared to infection of CDF macrophages

(expressing NLRP Iarianl) with the same Type II strain (Figure 5A).'After LPS-priming, high

levels of IL-1p and IL-18 secretion also correlated with NLRP1 sequence and macrophage

sensitivity to rapid lysis (Figure 5B, 5C). Furthermore, the HXBI (NLRPlvariant5), HXB15 and

HXB29 (NLRP 1 varianti) RI strains also produced IL-10 after infection in a manner correlated with

NLRP 1 sequence and macrophage sensitivity to Toxoplasma (Figure 5D). No IL-i IP or IL- 18

release was measurable from uninfected controls at any time point for any of the experiments

shown in Figures 5A-D (data not shown). If parasites were treated with Mycalolide B, there was

a significant reduction in cytokine production (Figure 5E) indicating that parasite invasion was

necessary for inflammasome activation. Finally, cleavage of IL- 10 and IL- 18 was detected in cell

lysates from LPS-primed, 76K or PRU-infected LEW, but not infected CDF and SD BMDMs,

and cleavage correlated with cytokine secretion (Figure 5F). Nigericin activation of the NLRP3

inflammasome in both Toxoplasma-sensitive (LEW, NLRP 1variants -expressing) and CDF or SD

(NLRPlvariantI-expressing) BMDMs confirmed previous findings that no general defect in the

caspase-1 pathway was present in rats (Figure S1, 5F) and (Newman et al. 2010)). Together

these findings indicate a perfect correlation between sensitivity to Toxoplasma-induced

macrophage cell death, decreased parasite proliferation, IL-1/IL-18 processing, rat resistance

97

to Toxoplasma infection and NLRP1 sequence (Newman et al. 2010), suggesting that

the Toxo] locus could be assigned to the Nlrpi gene.

No LPS* CDF76K* LEW 76K

LEW PRU+ CDP PRU

10 20Time post infection (hrs)

30

B2500 +s

|+LPS| BN2000 -CDF

E50 LEW1500SHR

1000

500 .

0 10 20 30Time post infection (hrs)

C

0

-9

+ LPS

1000

LEWV

500

0 2 4 6Time post inection (hrs)

+ L PS

HXB10 HXB15& HXB29

E low

S500.-j

0 10 20 30Time post infection (hrs)

LEW-4.

- - - - + +

SD

- 0

Figure 5. NLRPI-variant dependentfrom LPS-primed (0.1 ptg/ml,following Toxoplasma infection (MOI

cytokineI hour)

cleavage and secretion. IL-18 (A, C) and IL-1P (B, D)(B, C, D) or unprimed (A) rat BMDMs

for 76K and 3 and 5 for PRU). All infections are with strain 76Kunless otherwise indicated with the additional exception that SD BMDMs in panel B were infected withPRU. Results shown are averages from three experiments with SD shown, except measurements for PRUinfections in panel A which are the averages of four experiments, two with MOI 3 and two with MOI 5and those for the RI rats, which are from two independent experiments (triplicate wells/experiment/timepoint). No IL-I of IL-18 release was measurable from uninfected controls at any time point for any of theexperiments in A-D. P-values in (A) comparing CDF and LEW groups in (A) and (C) are <0.00 1 by two-way ANOVA. In (B) and (D), all P-values comparing NLRPI vananO expressing strains to theNLRP1 variand,"-expressing strains are <0.001 in all comparison combinations, by two-way ANOVA (E)

IL-l measurements from LPS-primed LEW BMDMs infected with Mycalolide-treated (3 PM, 15minutes) RH tachyzoites (MOI 1) after 24 hours; P-value comparing Mycalolide group to untreated is

98

A200

150.

R100

50

0

D800-

E 600

400.

S200

-T

.liftNT Myca-4B

FILIP is8

CDF LE COF LEWL PS141G

PRU

17kD

1 7k0

3

0.0024 (F) Western blot analyses for IL-18 and IL-1 in cell lysates and culture supernatants (indicatedby "S") of 76K-infected CDF and LEW BMDMs (MOI 3, 4 hours)(left panels) or PRU infected LEW andSD BMDM cell lysates (MOI 3, 24 hours)(right panels). NLRP3 agonist nigericin (40 pM, 4 hours) wasused as a positive control for inflammasome activation in the gel shown on the right. In the left pair ofgels, supernatants (no concentration, mixed 1:1 with SDS loading buffer) were loaded and Westerns werevisualized using IR-dye conjugated secondary antibodies and the LiCOR Odyssey. Cell lysates were alsorun, with processed IL-1p and IL- 18 shown with arrowheads in these gels, and pro-forms shown by redarrow. In the right gel, cell lysates are shown in Westerns visualized by chemiluminescence using acharge-coupled device camera. The unprocessed form of IL-Is is shown as the 37-kD band, and themature form is labeled 17 kD.

Nlrpi knockdown provides protection against Toxoplasma-induced pyroptosis

We utilized two methods to knock down expression of rat Nlrpi (designated as Nlrpla in

the rat genome) to determine if NLRP1 mediates Toxoplasma-induced rat macrophage

pyroptosis. First, an siRNA nucleofection approach was utilized. Only 20-35% of rat BMDMs

can be transfected with this method, as assessed by control nucleofections with GFP expression

vector and confirmed in parallel nucleofections in our current studies (data not shown). We

found that there was a significant protection against LEW macrophage death in cells transfected

with Nlrpi siRNA, compared to control siRNA, under conditions where 100% of BMDMs

succumbed (Figure 6A and 6B). The 20-30% difference in viability was correlated with the

number of successfully transfected cells, as reflected by the all-or-none nature of the protection

in individual cells assessed by microscopy (Figure 6A, inset). Surviving LEW BMDMs

remaining attached after longer periods of infection were verified to contain dividing GFP-

expressing Toxoplasma gondii by fluorescence microscopy (Figure 6C, D), and viability was

verified by MTT-staining (Figure 6D, left panel). Nonsurviving cells were completely detached

from monolayers. A second method of knockdown by lentiviral delivery of a homologous mouse

Nlrplb shRNA was used to achieve a 2.2-fold reduction in NlrpJ expression compared to

controls infected with a scrambled shRNA. Expression of Nlrpi was assessed by qPCR and

standardized against actin levels (Figure 6E). Knockdown correlated with increased parasite

99

proliferation and a higher number of vacuoles with more than one parasite (-60%), compared to

the macrophages treated with a scrambled control (35%) (Figure 6F). Host cell viability was

also increased by 30% in the shRNA knockdown condition (Figure 6G).

100

A30-

-20-

10-

n

B

C IE 2.5 0 CR

0 NIrP1o2.0-

1.5-

1.0-

0.5-

0.0

F 8

S60-

40-

- 20-

50-

40-

30-

20-

10-

A

Dr

# 4

M CRC Nirp1

'f

G

tIParasites/vacuole

H

100

5 o* 0

W LU WU-

Wj a_jU Q.

fO

LEWVECCDFVECLEWLWLEWCDFCDF 1ECDFCDF

100-80-

60-

40-

20-

0-

I

CR

0 CRC Nirp1

I-1

CDFCDF

Figure 6. NIrpI knockdown provides protection against Toxoplasma-induced pyroptosis andoverexpression of NLRPI vaants sensitizes resistant macrophages. (A) Viability of LEW BMDMsnucleofected with Nirpi siRNA pool or control siRNA (CR) 24 hours or 48 hours prior to infection withPRU (MOI 3) as measured by MTT assay at 5 hours post infection. Average from 6 separatenucleofection experiments (24 hours, n = 3, 48 hours, n = 3) are shown (triplicatewells/condition/experiment). P-values comparing N/rpl siRNA to controls is <0.001. Microscopy imagesof MTT stained nucleofected cells from representative 24 hours and 48 hours knockdown experiments arealso shown. (B) Viability of LEW BMDMs nucleofected with Nirpi siRNA pool or control siRNA (CR)36 hours prior to infection with PRU (MOI I) as measured by MTT signal at 24 hours post-infection.Average of 4 separate nucleofections are shown (triplicate wells/condition/nucleofection experiment) (C,

101

LEWLEW LEWCDF CDFLEW

--

I

4,1' 24 NRIIPI(48h)

-4CR _T_

I '

1,

D) Toxoplasma division in individually surviving nucleofected LEW BMDMs from (B) at 24 hours post-infection. In C cells were fixed prior to microscopy, while in D cells were MTT-stained and fluorescencemicroscopy performed with no fixing. Note that all non-transfected or control siRNA transfected LEWmacrophages which have succumbed are not present in these fields (detached by 24 hours), while theMTT-negative ghosts and organelles of these lysed cells can be seen in parallel experiments at the earlier5-6 hour time points, as shown in panel A. (E-G) Knockdown by the alternative lentiviral shRNAmethod was confirmed in LEW BMDMs by qPCR (E) and parasites per vacuole counts (F) and viabilityby MTS assay (G) were assessed in NlrpJ-knockdown LEW BMDMs after RH strain infection (MOI0.5). P-values by t-test comparing knockdown to controls is 0.03 for C and 0.01 for D. (H) Viability ofLEW and CDF BMDMs nucleofected with full length HA-tagged NLRP 1 constructs at 24 hours prior toinfection with PRU (MOI 5) was measured by MTT assay at 5 hours post-infection. Cell lysates fromnucleofected cells were made at 32 hours post-transfection and analyzed by Western using anti-HAantibody. Superscripts indicate the NLRP1 construct or vector that was transfected into the cell. Graphshows average from two nucleofection studies, with duplicate wells/condition/experiment. Lysates arefrom one of these nucleofections. There is no significant difference between any of the nucleofected LEWcells. The P-value comparing the CDF cells (expressing NLRPlvafan) transfected with LEW(NLRPlvafian) to CDF cells nucleofected with vector or CDF (NLRPlvaan ) is <0.0005. Presence ofMTT-negative cells was also verified by microscopy for each well. Similar data is also shown in FigureS8, with anthrax LT control treatments. (I) Representative microscopy images of MTT viability stainingfor LEW and CDF BMDMs nucleofected with full length HA-tagged NLRP1 constructs 36 hours prior toinfection with PRU (MOI 3) or treatment with LT (PA + LF, each at 1 pg/ml). MTT staining wasperformed on Toxoplasma-infected cells at 8 hours post-infection and on LT-treated cells at 5 hours post-infection. Superscripts indicate the NLRP 1 construct or vector that was transfected into the cell.

Overexpression of NLRPvariant 5 sensitizes CDF BMDMs, but not fibroblasts and mouse

macrophages, to Toxoplasma-induced pyroptosis

We next overexpressed HA-tagged NRLP Ivaian2 and NLRP 1 variant 5 constructs (Levinsohn

et al. 2012) in rat BMDMs by nucleofection to test if this alters susceptibility to parasite-induced

pyroptosis. The efficiency of transfection ranged from 25-40% in BMDMs in individual

nucleofections (as assessed by monitoring of a co-transfected GFP construct in control cells).

The LEW BMDMs did not gain resistance when transfected with the resistant CDF

NLRP I vaiant2, but were sensitized to treatment with anthrax LT, confirming expression of the

CDF NLRP Ivanan in a subpopulation of nucleofected cells (Figure S7). There was a significant

sensitization to parasite-induced pyroptosis in CDF cells transfected with the LEW

NLRPvariants (Figure 6H, Figure S7), while these cells remained almost 100% susceptible to

102

rapid lysis by LT (Figure S8). Microscopy confirmed cell death for both Toxoplasma-infected

CDF cells expressing LEW NLRPvarian ts and LT-treated LEW cells expressing the CDF

NLRPI variant2 (Figure 61). These results confirm that the LEW NLRPlvarianl -mediated

sensitivity to Toxoplasma is dominant, much in the manner the resistance of LEW rats to the

parasite was previously shown to be a dominant trait (Cavailles et al. 2006). They also re-

confirm that the sensitivity to anthrax LT, mediated by the CDF NLRP Ivariant2 is a dominant trait.

Interestingly, fibroblast HT1080 lines expressing these rat NLRP1 constructs (Levinsohn et al.

2012) were not sensitized to Toxoplasma-induced pyroptosis even when transiently transfected

and confirmed to express caspase-1 along with NLRP1 (Figure S8, panel A). These results

confirmed that a macrophage cofactor or the macrophage cellular environment is required for

parasite-induced pyroptosis. Furthermore, infection of mouse macrophage cell lines stably

expressing rat NLRP1 constructs also did not result in sensitization to Toxoplasma (Figure S8,

panel B), suggesting the presence of other factors in murine macrophages, or the BMAJ

macrophage cell line, that result in a dominant resistance to pyroptosis or the absence of a factor

needed for interaction with rat NLRP1 and subsequent pyroptosis. All tested mouse macrophages

from any inbred strain, to date, have been resistant to Toxoplasma-induced pyroptosis (data not

shown and Figure S8, panel C). The competition of endogenous murine NLRPla and NLRPlb

proteins for co-factors required for pyroptosis in the mouse macrophage may explain this

resistance.

Together, the results presented in this work indicate that NlrpJ expression contributes to

the ability of BMDMs from rats resistant to Toxoplasma infection to control parasite replication,

most likely because of its role in mediating Toxoplasma-induced macrophage pyroptosis.

103

Discussion

The Toxol locus that controls rat susceptibility to toxoplasmosis (Cavailles et al. 2006)

was previously mapped to a region of rat chromosome 10 containing the inflammasome

sensor Nlrpi. In this work we identify Toxoplasma as a novel pathogen activator of the NLRP 1

inflammasome. Until this work, anthrax LT was the only known activator of this inflammasome

sensor (Newman et al. 2010; Levinsohn et al. 2012; Hellmich et al. 2012). We now demonstrate

that like LT, rapid Toxoplasma-induced rat macrophage cell death is a pyroptotic event for which

sensitivity correlates to NLRP1 sequence. Type I, Type II and a variety of genetically diverse T.

gondii strains induce rapid pyroptosis in macrophages derived from inbred rats expressing

NLRP1 variants, while macrophages from BMDMs expressing NLRP 1 variant,2 are resistant to the

parasite. This is the inverse of what is known for LT, where NLRPIvariant 1,2 confers

sensitivity (Newman et al. 2010). In rats, macrophage sensitivity to Toxoplasma-induced cell

death inversely correlates with whole animal resistance to infection. Rat strains historically

susceptible to chronic Toxoplasma infection (e.g., CDF, BN, SD; NLRPIvariantl,2) have

pyroptosis-resistant macrophages whereas resistant rats that cure infection (e.g., LEW, SHR;

NLRPlvariants) harbor macrophages that undergo parasite-induced pyroptosis. This suggests that

the ability of the macrophage to allow parasite proliferation and possibly dissemination is linked

to resistance to parasite-induced macrophage pyroptosis. Similar findings were previously

described for mouse Nlrplb-mediated control of anthrax infection. Mice resistant to Bacillus

anthracis have macrophages expressing Nlrplb variants which confer macrophage sensitivity to

anthrax LT, and resistance is linked to the IL-Ip response induced by toxin (Moayeri et al. 2010;

Terra et al. 2011). The idea of control of parasite proliferation at the macrophage level is

supported by findings that macrophages are among the first cell types to be infected when an

104

animal ingests Toxoplasma cysts or oocysts (Mordue and Sibley 2003; Suzuki et al. 2005) and

innate immune cells are used to traffic from the site of infection to distant sites such as the brain

(Lambert and Barragan 2010).

In parallel to the consequences for parasite proliferation after NLRP 1 activation, the pro-

inflammatory cytokines, IL-ip and IL-18, which are substrates of caspase-1, are cleaved and

released following inflammasome activation. We demonstrate that these events only take place

after infection of pyroptosis-sensitive macrophages in a manner correlating with NLRP1

sequence. It is possible that the release of these cytokines of the innate immune system could

also play a role in controlling toxoplasmosis. IL-18 was at one time known as "IFN-inducing

factor" and the role of IFN-y in resistance to Toxoplasma is extensively documented (Melo,

Jensen, and Saeij 2011; Hunter and Sibley 2012). Treatment of resistant LEW rats with anti-IFN-

y antibodies does not reverse resistance but results in a much stronger antibody response, while

anti-IFN-y antibody treatment in susceptible rats causes an increase in parasite burden (Sergent

et al. 2005). Altogether these findings suggest that IL-18, (through actions by IFN-y) could be

important for inhibition of Toxoplasma replication in rats, but that the cytokine's actions do not

necessarily prevent parasite dissemination. On the other hand, it is important to note that

as Toxoplasma can replicate and form cysts in many cell types that do not undergo pyroptosis,

macrophage death may play a role strictly in dissemination. Thus, we suggest the combined

consequences of inflammasome activation, macrophage cell death and IL-I/IL-18 secretion, on

both dissemination and parasite proliferation, may ultimately result in resistance to Toxoplasma.

The only difference between the NLRP1 proteins from Toxoplasma-resistant

and Toxoplasma-sensitive inbred strains is an 8 aa polymorphic region in the N-terminus of the

protein, in a region of unknown function (Newman et al. 2010). LT cleaves

105

NLRPI variant],2 proteins to activate this sensor and induce pyroptosis, while NLRP 1variant5 is

resistant to cleavage (Levinsohn et al. 2012). How Toxoplasma activation of NLRP1 varies

between rat strains based on an 8 aa sequence difference is unclear. The similar induction of

pyroptosis we observed with numerous Toxoplasma strains suggests that the factor activating

NLRP1 is unlikely to be parasite strain specific, or at least is conserved among multiple strains.

One logical hypothesis is that the parasite-encoded effector molecule responsible for activation

of NLRP1 is, like LT, a protease, but one which targets the LT-cleavage resistant sequence found

in NLRPlvariant5. Toxoplasma secretes a large number of proteases (Choi, Nam, and Youn 1989;

Dou and Carruthers 2011; Dou, Coppens, and Carruthers 2013; Kim 2004; Shea et al. 2007). It is

unlikely that such a secreted protease could be derived from the rhoptries, because rhoptry

secretion into the host cell was not sufficient to induce cell death. To date, we have been unable

to observe any cleavage of NLRP1 in Toxoplasma infected fibroblasts, which overexpress an

HA-tagged variant of the protein (data not shown). It has also been recently shown

that Toxoplasma can secrete effectors post invasion beyond the parasitophorous vacuole

membrane (Bougdour et al. 2013) and these could be candidate effectors for NLRP 1 activation.

An alternative hypothesis to the parasite causing direct cleavage of NLRP1 is that the N-terminal

polymorphic region of rat NLRP1 affects this protein's interaction with a different host 'sensor'

acting as adaptor for the inflammasome, much in the manner described for the

NLRC4/NAIP5/NAIP6 inflammasome recognition of flagellin (Kofoed and Vance 2011; Zhao et

al. 2011). This unknown adaptor would interact with Toxoplasma or its effectors in all

macrophages but may be limited by its ability to interact with the N-terminus of

NLRPlvarian, in rat BMDMs, or alternatively it could act as a direct inhibitor with specificity

for these variants. The likelihood of a proteolytic activation of NLRPI is also reduced when

106

considering the finding that mouse ortholog NLRPIb proteins harbor an LT-cleavage site similar

to rat proteins (Hellmich et al. 2012) but are highly resistant to Toxoplasma-induced pyroptosis

in a manner independent of NLRPlb sequence or LT sensitivity (Figure S8). Furthermore,

mouse macrophages could not be sensitized by rat NLRP1 overexpression. This finding was in

contrast to the sensitization of the same cells to LT-mediated cell death (Levinsohn et al. 2012),

suggesting resistance of mouse macrophages to Toxoplasma-induced pyroptosis was dominant to

any NLRP1-mediated effect, or (less likely) that co-factors required for parasite-mediated

activation were only present in rat cells. Alternatively, the endogenous Toxoplasma non-

responsive NLRPla and NLRPlb proteins in mouse macrophages could compete in a dominant

manner with expressed rat NLRP1 for co-factors required for pyroptosis. Interestingly, human

NLRPI does not contain an LT cleavage site in its N-terminus (Moayeri, Sastalla, and Leppla

2012). Instead human NLRP 1 contains a pyrin domain required for association with the adaptor

protein ASC (Faustin et al. 2007), which does not appear to play a role in NLRP1-mediated

rodent cell death (Nour et al. 2009; Broz et al. 2010). SNPs prevalent in this N-terminal region of

human NLRP 1 have been correlated with the severity of human congenital

toxoplasmosis (Witola et al. 2011). In those studies, knockdown of NLRP1 in human monocytic

lines led to reduced cell viability after Toxoplasma infection, perhaps by allowing uncontrolled

division of the parasite. Unlike our findings in rat cells, a protective role for human NLRP1

against macrophage death was suggested. It seems likely that the cell death observed in these

human cell studies, which occurred over a period of days, differs from NLRP 1-mediated rapid

pyroptosis of rat cells, which occurs over a period of hours. Future studies are required to

determine the mechanism of NLRPI action in human cells.

107

In summary, we have established that Toxoplasma gondii is a new activator for the NLRP 1

inflammasome. The identification of T. gondii as the second pathogen to activate the NLRP1

inflammasome raises the question whether this parasite activates the sensor via a novel

mechanism, or whether proteolytic cleavage is required, in a manner similar to anthrax LT.

108

Materials and Methods

Ethics statement

All animal experiments were performed in strict accordance with guidelines from the

NIH and the Animal Welfare Act, approved by the Animal Care and Use Committee of the

National Institute of Allergy and Infectious Diseases, National Institutes of Health (approved

protocols LPD-8E and LPD-22E) and the MIT Committee on Animal Care (assurance number

A-3125-O1).

Materials

Ultra-pure lipopolysaccharide (LPS), nigericin (Calbiochem/EMD Biosciences, San

Diego, CA and Invivogen, San Diego, CA), 3-(4,5-dimethyl-2-thiazolyl)-2,5-dipheny

Itetrazolium bromide (MTT) (Sigma, St Louis, MO), Mycalolide B (Wako USA, Richmond,

VA) were purchased. LT consists of two polypeptides, protective antigen (PA) and lethal factor

(LF). Endotoxin-free LF and PA were purified from B. anthracis as previously described (Park

and Leppla 2000). Concentrations of LT refer to equal concentrations of PA+ LF (ie, LT 1 pg/mi

is LF+PA, each at 1 pg/ml).

Rats

Brown Norway (BN/Crl; BN), Fischer CDF (F344/DuCrl; CDF), Lewis (LEW/Crl;

LEW), Spontaneously Hypertensive Rat (SHR/NCrl; SHR) and Sprague Dawley (SD) rats (8-12

weeks old) were purchased from Charles River Laboratories (Wilmington, MA) and used as

109

source of bone marrow. Certain experiments utilized F344/NTac rats from Taconic Farms

(Germantown, NY). The recombinant inbred (RI) rat strains HXB1, HXB15 and HXB29 are

derived from the progenitor strains BN-Lx and SHR/Ola (Pravenec et al. 1996; Pravenec et al.

1989; Printz et al. 2003). The microsatellite marker genotypes and linkage maps used in mapping

LT sensitivity using the HXB/BXH RI collection have been described (Newman, Printz, et al.

201 Ob).

Parasites

Tachyzoites from Type I (RH) and Type 11 (76K or Prugniaud [PRU]) strains expressing

luciferase and GFP from the plasmid pDHFR-Luc-GFP gene cassette (Saeij et al. 2005) were

used for most experiments. The following strains (haplogroup/type in parentheses) were used in

a survey of effects on rat macrophages: GT1 (I), ME49 (II), DEG (II), CEP (III), VEG (III),

CASTELLS (IV), MAS (IV), GUY-KOE (V), GUY-MAT (V), RUB (V), BOF (VI), GPHT(VI),

CAST (VII), P89 (IX), GUY-DOS (X), VAND (X), Cougar (XI), RAY (XII), WTD3 (XII). All

parasite strains were routinely passaged in vitro in monolayers of human foreskin fibroblasts

(HFFs) at 37'C in the presence of 5% CO2, spun and washed prior to quantification by

hemocytometer counts. In some experiments, Mycalolide B (3 IM, 15 minutes) or DMSO was

used to pretreat isolated parasites prior to washing in PBS (3x) before infections. The viability of

these Mycalolide B- or DMSO-treated parasites was assessed in each experiment by adding them

to a monolayer of HFFs and staining for STAT6 activation induced by the parasite secreted

rhoptry kinase ROP 16. Mycalolide B-treated parasites were able to secrete ROP 16 but could no

longer invade. In other experiments parasites were lysed using cell lysis solution (Abcam,

110

Cambridge, MA) to assess LDH activity. Parasite viability and health differed from experiment

to experiment, accounting for variations in experimental results that are reflected in standard

deviations for pooled studies.

Cell culture, nucleofection, toxicity, cytokine measurement, Western and microscopy

studies

BMDMs were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented

with 30-33% L929 cell supernatants as previously described (Newman et al. 2010; Wickliffe,

Leppla, and Moayeri 2008), or with minor modification (20% fetal bovine serum, 50 pLg/ml

penicillin and 50 pg/ml streptomycin). NLRP1-expressing HT1080 or macrophage BMAJ lines

and their growth conditions have been previously described (Levinsohn et al. 2012) . The c-myc

tagged rat caspase-1 gene was synthesized by GeneArt (Regensburg, Germany) and cloned into

pcDNA(3. 1)+ vector for expression in HT1080 cells by transfection with TurboFect (Fermentas,

Glen Burnie, MD) using manufacturer's protocols. HA-tagged LEW and CDF NLRP1 expressing

constructs used in BMDM nucleofection experiments have been described (Levinsohn et al.

2012c). Endotoxin-free control vector or various NLRPI expressing constructs were purified

(Endofree kit, Qiagen, Germantown, MD) and nucleofected (1.2-3.0

g/x 1O6cells/nucleofection) into rat BMDMs using the Amaxa Nucleofector (Lonza,

Walkersville, MD) (kit VPA-1009, program Y-001). Nucleofections were performed at -24,

-36, -48, and -72 hours prior to infections with parasite. Toxicity and viability assays were

modified from previously described methods (Newman et al. 2010; Wickliffe, Leppla, and

Moayeri 2008). Briefly, animal-derived BMDMs with or without LPS priming 0.1 ptg/ml, 1 h)

111

were infected with Toxoplasma at various multiplicities of infection (MOIs) or treated with

anthrax LT (1 pig/ml) and cell viability was assessed at different time points by one of three

methods. 1) MTT staining (0.5 mg/ml) was performed as previously described (Newman et al.

2010; Wickliffe, Leppla, and Moayeri 2008); 2) MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carb

oxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)was used to measure viability with the

CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) according

to manufacturer protocol ; 3) Lactate dehydrogenase (LDH) release assays were performed in

select experiments according to manufacturer protocol (Roche Diagnostics, Mannheim,

Germany). For luciferase assays, cells were lysed in 1 x Lysis Reagent (Promega) and luciferin

(Caliper Life Sciences, Hopkinton, MA) added prior to luciferase activity readings. In all

experiments culture supernatants were removed for cytokine measurements by ELISA (R&D

Systems, Minneapolis, MN and Abnova Corporation, Walnut, CA) or Western blotting, with or

without concentration using Amicon filters (3000 Molecular weight cutoff) (Millipore, Billerica,

MA). Cell lysates were made from infected cells as previously described (Newman et al. 2010;

Wickliffe, Leppla, and Moayeri 2008). Anti-rat IL- 1 P (Abcam or Santa Cruz BT, Santa Cruz,

CA), anti-rat IL-18 (Santa Cruz BT) or anti-HA antibody (Roche Diagnostics) were used as

primary antibodies. Secondary IR-dye conjugated or HRP-conjugated antibodies were from

Rockland (Gilbertsville, PA), Licor Biosciences (Lincoln, NE) or Jackson Immunoresearch

(West Grove, PA). Immun-Star Western C substrate (BioRad, Hercules, CA) and a charge-

coupled device camera (Chemidoc XRS, Biorad) or the Odyssey Infrared Imaging System (Licor

Biosciences) was used for Western visualization depending on the secondary antibody used for

detection. For select microscopy studies phase contrast images of MTT-stained cells were

acquired on a Nikon Eclipse TE2000-U microscope without cell fixation followed by

112

fluorescence image collection for the same field. For other fluorescence microscopy studies

nucleofected cells were plated on poly-lysine (Sigma, St. Louis, MO) treated coverslips prior to

infection and fixed (4% paraformaldyde, Electron Microscopy Sciences, Hatfield, PA), with or

without permeabilization (0.1% TritonX-100). Immunostaining was with anti-HA antibody

(Roche Diagnostics) and Alexa Fluor 594 secondary antibody (Invitrogen). For

immunofluorescence staining of surface antigen (SAG)-1 or assessment of STAT6

phosphorylation, cells were fixed (3% formaldehyde) and permeabilized (0.2% TritonX-100 or

100% ethanol) followed by staining with a rabbit polyclonal antibody against human pSTAT6

(Santa Cruz BT, Santa Cruz, CA) or rabbit polyclonal antibody against Toxoplasma surface

antigen (SAG)-1. Alexa Fluor 594 secondary antibodies were used for detection as has been

described (Rosowski and Saeij 2012).

RNA knockdown studies

NLRP1 knockdown was achieved by two methods. First, siGENOME SMARTpool

siRNA set of four, targeting rat Nlrpla (D-983968-17, D-983968-04, D-983968-03, D-983968-

02; target sequences

of GGUCUGAACAUAUAAGCGA, CCACGGUGUUCCAGACAAA, GCAUUACGUUCUCU

CAUGU, GCAGUACGCAGUCUCUGUA) and siGENOME non-targeting siRNA pool (D-

001206-14-05, target sequences

of UAAGGCUAUGAAGAGAUAC, AUGUAUUGGCCUGUAUUAG, AUGAACGUGAAUU

GCUCAA, UGGUUUACAUGUCGACUAA) were obtained from Thermo Sciences-Dharmacon

(Pittburgh PA). siRNA pools were nucleofected (200 nM) into rat BMDMs (day 5 or 6 of

113

differentiation) using the Amaxa Nucleofector (Lonza, Walkersville, MD) (kit VPA-1009,

program Y-001) at -24, -36, -48, and -72 hours prior to infection. Alternatively, on day 2 of

differentiation BMDMs were infected with high-titer lentivirus (Broad Institute RNAi

consortium) encoding shRNA against target

sequence TGATCTACTATCGAGTCAATCdesigned against murine Nlrpib with high

homology (18 out of 21 nucleotides, perfect seed sequence identity) to rat Nlrpla or the control

shRNA with sequence

(GCTTATGTCGAATGATAGCAA or GTCGGCTTACGGCGGTGATTT). Puromycin

selection (6 pg/ml) of lentivirus infected cells, followed by qPCR analysis (Nlrpla primers

were 5'CATGTGATTTGGACCTGACG'3, 5'TCTTTGCCTGCAAGTTTCCT'3, actin primers

were 5'GTCGTACCACTGGCATTGTG'3, 5'CTCTCAGCTGTGGTGGTGAA'3) verified

knockdown. Expression of Nlrpla was normalized against actin expression levels.

Whole transcriptome sequencing and SNP analyses

SNP and haplotype analyses for the HXB, SHR, F334 and LEW rats were performed based

on data and genome analysis tools at the Rat Genome Database (RGD), Rat Genome Database

Web Site, Medical College of Wisconsin, Milwaukee, Wisconsin (http://rgd.mcw.edu/). Any

gene within the region fine mapped using the above haplotype analysis that contained at least

one non-synonymous SNP was identified using Ensembl's Biomart engine and the rat short

variation (SNPs and indels) (Rnor_5.0) dataset. We then used the variant distribution tool on the

RGD website to identify which SHR strain genes contained at least one SNP difference from

F344 and BN strains. Nucleotide positions correspond to the RGSC3.4 assembly. Further fine

114

mapping analyses were performed by whole transcriptome sequencing and novel SNP

identification. RNA (Qiagen RNeasy Plus kit) was isolated from unprimed and LPS-primed (100

ng/ml) LEW and SD BMDMs or LPS-primed BN BMDMs. mRNA purified by polyA-tail

enrichment (Dynabeads mRNA Purification Kit, Invitrogen) was fragmented into 200-400 bp,

and reverse transcribed into cDNA before Illumina sequencing adapters (Illumina, San Diego,

CA) were added to each end. Libraries were barcoded, multiplexed into 5 samples per

sequencing lane in the Illumina HiSeq 2000, and sequenced from both ends (60 bp reads after

discarding the barcodes). Sequences were mapped to the Rat genome (rn4) using Bowtie

(2.0.2) (Langmead et al. 2009) and Tophat (v2.0.4) (Trapnell et al. 2012). To identify SNPs from

the RNAseq data in the interval fine mapped above, Bam files were processed with samtools

(0.1.16, r963:234) mpileup function, with m4 as reference sequence. Read pileups were

processed across all five samples using VarScan.v2.2.11 and the mpileup2snp function

(parameters: -min-coverage 2 -min-reads2 1 -min-var-freq 0.01 -p-value 0.05 -variants).

Resulting variant positions were annotated using UCSC Genome Browser's "Variant Annotation

Integrator". SNPs identified between 5 samples (2 SD, 2 LEW, 1 BN) were filtered for

concordance and homozygosity between the two independent LEW samples and BN having the

same nucleotide as the reference genome (which is from BN), and subsequently filtered for non-

synonymous SNPs where LEW differed from BN and SD. It should be noted that not all known

LEW SNPs in Nlrpi are discovered using this procedure as the N-terminal NLRP1 region

contains a stretch of eight amino acids that differ between LEW and BN and our procedure for

mapping reads to the genome does not allow for that many mismatches. Similar problems lead to

underreported Nlrpi SNPs in the RGD website.

115

Supplementary Data

A

0100-

80-C

60-

o 40-

20-

0.

0C,

B

\ N0

'N%IR

* Y" X

0U,(U

0

a:I0-4

100'

80.

60-

40-

20-

0.

00

.a

C -LPSW +LPS

100

I fi

50

0 bLo

01 ,I-

D

C=.Ir-j

2000-

1000-

~ ~-- --

N

0

M +LPS

-y N - - -

0A Atq~~

Supplementary Figure SI. Parasite-derived MTT signal and LDH levels.(A) CDF BMDMs wereinfected PRU (MOI I or 3) and MTT assessed at 6 hours post-infection relative to uninfected controls (B)

RH parasites at shown MOI were lysed in the absence of cells using the same volume to lyse uninfected

BMDM monolayer used in typical experiments and LDH levels measured (C, D) Primed or unprimed

(LPS 100 ng/ml, 2 hours) LEW BMDMs were infected with RIH (MOI 0.5 or 1.0, as indicated) or treated

with LEW macrophages or HFFs that had been syringe-lysed and prepared in parallel to parasites. Thevolume of cell lysates added to LEW BMDMs is equivalent to the volume of parasites added at the MOI

indicated in parentheses. Viability and IL-1I3 release were then assessed 24 hours post infection.

116

I-F-

I

5

I

LEW CDF LEW CDF+ + + +

+ +- -

- - + +

SD SD SD+ + +

+

- + +

j *ii - 37kDa

< 17kDa

Supplementary Figure S2. Activation of the NLRP3 inflammasome by nigericin in CDF and LEW

rats. CDF or LEW BMDMs were pre-treated with LPS (1 pg/ml, 2 hours) followed by either LT (1 tg/ml

LF+I pg/ml PA, 90 minutes) or nigericin (10 pM, 1 hour). In a separate experiment, SD BMDMs were

LPS treated (100 ng/ml, 2 hours) and either infected with RH strain (MOI 0.5, 6 or 8 hours), or treated

with nigericin (40 pM, 4 hours). Supernatants were Amicon-concentrated prior to Western blotting. The

unprocessed form of IL-I P is 37 kD. The mature cleaved form is 17 kD.

117

LPSLT

NIGTOXO -

-A ' - Varmnfto, ANIMMUft-

]Animnal SENSITVITY TO TOXOPLASMOSIS S S S |RMacrophage SENSITIVITY TO PYROPTOSIS R R R S'__________f7I

CHR 10 (Mbp;ALLELE INCUEUCL

TACAC;TC TTCT CGCGACCTCATTAATTA - CTG{:MG~GAACGCTGGTTTCGTTC-_CCCCCT CCAG AAGGGAATT.TCCGGGACTGC-CCfi.CTTGG3AITT-CTTCCTT2CGCGAT3GC-TCAGAAGCTC

'ACA TCA3TCACGGATCATGTCTCCCTCACACCGCCCGG'TAGGGTCACAGTG GTTCTCCCTTGCAGACCG

AATMGACTACCTG;CATAGGGCA AAATTCACCAT. rTGAATTT GTGGCACAG7GTA7AG G"AAT ACCCAAACACTTC CT-G-CCAAAAGAAAAAG-GACCTC.ITCAAAGTTTTTTC C TA GCGTGCACCTTGG2AGTIGTGA

ACC'CCGATGGTAAAGCCACACATGTCCCACAATA-GC T CIGAACAGGATT CCA'TCC-GACAGCC AC3ACGAAA

ICCNCNP 2A ACI.353IIG AACAGGAACACATCCC CACGAGGCCA3CGI[.3G3CC AGACGAACTGAGAC GCTGCCGGGTAAGAT ACCCCNP2C3,AA 554113CC C AG AGCC13CTITCTT 3TACCCCTl'CA&TCCCCCGCTJCCCCAGTCTTICCCCGCITCGCCCC3G>CAACC3__

rsT-CTCACAG C 1Cc. TACCACACC GTCTAC MAA_ CC CCGCIGTCTTCTC TACT.:ATA

6 3513 JG r T G11, CACA ATTA T i T R I fAAi CC IAT A 'CC A 'CCCcC

- _ CC1 . 55: 2 AG GAM C G AM AA . . > A XA TA G AIA C, A.'ICA _ 1 _3 A 1

t_______e A ,CIIAC.GCA.I-CC, .3x ,.>. Ic , ATlfIC,, A f Vr.' CA pAT

AICCIT<I GA A t_ 1__ 'AC T _, .'ATGGCCCCOMMT JG22_ _A, T

.CCCCCCP23CACC -. 31ICICC CC CC.ACC , lMCACATTT'CC TTC"T'CCAG:C CM CCGCACTGCACCA 'G' CC ,A ''3,TGC AGC

- C .Mod32CG7 GAA AG7GC y-7:aG TAGAAGM GCC..3-, :T TGGCTATTAG-,AP AGCO G GT- O ~ ~ 7W !a 704t AG 7MTM GTAG3TACG M AGCG TK2GT1-,'AG.AGC,'1 ACCINC~ 1- - A.T TG

'&", 7,E 57077 |A.:C6 C GCA T c TCMAa A T r_ 'Ann 'C TC TAS G /TA T C C TG AA

G~~omom ~ MC9 kA. TTACCC 7A All;CC T.-CITCTC f-TiA 7C C G AC ATGA--CA CACTTGG TAC

iN,_ s;14. 7 ' 124C AG TA I A'ATA~ 1TA3 rAa !.-,AT TGTGCCTGG GTC AGT AA

2: !M 575461-C GIA AGGA ~ % G rGCCA C.--I~'% _T2 A^TGGl:CK AF CG __C iG_S 5853TfC G -CA ,mACZCTAC' A~CAGOG CGTACCSGGA GSC AAAICT TT

EOM NP27,1,ET, T 5I5EWt CIT GAGGAGCCGGAGAGTAA GCGGC GC AACAGCAAGGGACAAGTGCG3A1_ G GTGTGTG C_CCCGCCCA.CCCCCCCCC-CCCMCCCCCCACSCTCCAGCGCTCCCCCCCAACAAA.CAC.CZA

tTGTGC A CAAG G ACCCACATG'A A .. ''AAAA.A AA

L .;C_; _ AGTG- r3'AACGTC""' T-__A .CCA AAG GMC AG ~--CC *A 3C C AA'CC CCCCCG o____7,C TTGAG,_AAtA Gt T f -CCATT7A -_,c--~iCM G T - CTGAAAGAA~G GC TT TT.1333C &T AITCCCCC AACC G-CA A

Cm! 121CA CAACCCCCACCTACAAT-C- C, IAC.',CA 'JA~q :-A , TG rG CG CCCA- 1G:' CCGG C C- AGCGGACAGGC G < AA

.ACC' AGAAC I GTICCICTGAGG.rGGAGCC3C'CA 1- A. CICC C3CCACACCI,G") Qt2 " C -TGCTO-TG--'TCAGG3,-A' :G TGCTSA ',-Ak--,TTAI :T CCATCCCAAACGC AAGAGCCCG [

TT7 7

AA

CC

AA__7AM AA

CC V'-

I I

Supplementary Figure S3. Fine-mapping of the Toxol region using whole transcriptome

sequencing, SNP and haplotype analyses. Table was generated using SNPlotyper tool at RGD.

Alternative SNP annotations can be found at that site. Shaded area indicates the new boundaries

for Toxol locus based on comparison of the inbred and RI rat strain SNP genotypes for the 7 rat strains

BN, F344 (CDF), LEW, SHR, HXBl, HX1B15 and HXB59.

118

INYMIJUL

- -_ .P36>'.

Rl S31 F344 LEW

AA AAAA AAcc cc

CC cc

CC cc

CC ccTT

GG GGlaG GG

_T

rAA G

_T

ERHC-NP2796609 's q 3 as1N1RN0-;NP27%E 12 53 107% 1

46t~m Im - 37.1El;'.& N _1 ~ 10ro

C3RtW-INP279 !a644a CIT

FRI r P

- - 54--23-- E-54%2725

C, M a-CCAGC 7TG 'C TMGAUCI TUAGCAGTAALA I C I- 7,- G F1ArT.A t T I - -- AAG~u s I A- t- -AAGATMGGGTAAGTAlG z'-CAAAT_ IA- T.ACAGA -CCT[GlrGGCTGAGGAAGC AACGGAAG;-TAGG

TAAMkG ~i AAT T:A ATCAAGTATGAAGGCG

Cl C-1

RSSHRGGAA_cccccc

cccc

GGGG

GG

GGTT

GGUGC4

T

cc

GG

AA

AAT

cc

GGGG

TT&AGG

cc-

__....

7'

A7 [TT TTCA AA AAAG IGG G

TC CC CCCT Tr TTTA AA AA

6C AG GG GGcc GC CC CCAA CA AA A

Subset of genes expressed in LEW BMDMs (+/- LPS) with non-synonymous SNPsDifferent in Different in

NS SNP SHR vs SDIBN vs.(All rats) F334/BN LEWIS

GENE Start End LEWIS-NS LEWIS-LPS SD-NS SD-LPS BN-LPS (Ensembi) (RGD) (RNAseq)Aurkb 55798709 55804367 204 4.2 158 9.4 11.8 YES YESNlur14 5674521 56757435 51 25 4.0 36 34 YES YES YES

z16 57296125 57307217 100.5 264.3 111.6 368.1 262-9 YES YES YES57963707 58007925 45 9.8 4.8 13.5 13.0 YES YES YES

Tp53 56399720 56411150 36.9 45.3 38.1 60.9 59.5 YES YES NI

DIg4 56864458 56890626 2.3 3.1 2.2 2.9 2.3 YES YES N,Arrt2 57244070 57284166 23.0 6.5 24 1 8.7 10,0 YES YES N 2Rabep1 57746746 57847498 9.5 181 76 12.0 11.1 YES YESRpi26 55662219 55665752 42.8 18.5 571 30.0 33.5 YESRGD1563106 55768181 55788836 48 25 5.4 2.2 24 YES NTrappcl 56117228 56118815 68.7 66.7 574 56.9 62.5 YES N<Lsmdl 56190118 56192730 50.7 27.4 45.0 26.6 33.8 YES NOEff4a1 56484231 56489739 348.4 422.1 335.2 312.3 307.2 YES NI_RnaseO 57077235 57078954 469.0 569.9 502.9 514.6 490.9 YESPsmb6 57370396 57372704 34.4 25.6 358 27.8 24.6 YESPfr.1 57531660 57534366 1123.6 1085.2 1133.8 10770 1134.6 YESKif1c 57580645 57622517 30.8 31.0 27.8 24.3 19.6 YESCIbp 57881782 57886434 113.0 72.1 964 877 97.2 YESTxndcl7 59100037 59103010 135.1 107.9 165.1 76.9 81.8 YESXAF1 59185149 59196549 8.4 248.9 7.4 252.9 216.8 YES

Supplementary Figure S4. Whole transcriptome analyses of LEW, SD and BN rats. Summary of

genes expressed in both LPS primed and unprimed conditions are shown for which non-synonymous

SNPs (NS) existed. For each SNP, comparison of Toxoplasma-resistant and Toxoplasma-sensitive rat

genotype correlation to phenotype was then used to narrow Toxo] to four candidates, in red.

119

Toxo Hoechst PSTAT6

-O

C)

Supplementary Figure S5. Parasites treated with Mycalolide B are able to secrete ROP16 and

induce activation of pSTAT6. HFFs were infected with GFP-expressing type I parasites that were

pretreated with 3 pM Mycalolide B or vehicle control for 15 minutes. Cells were infected for four hours

and then fixed with 3% formaldehyde, permeabilized with 100% ethanol and blocked. A rabbit antibody

against human pSTAT6 was used as the primary antibody, followed by a goat- anti-rabbit antibody

conjugated to Alexa Fluor 594. Green = Parasite, Blue/Pink = Hoechst, Red = p-STAT6.

120

N

A B DNA

X

100

S80

-060

40 T2 0

Sprague -Dawley Lewis

Supplementary Figure S6. Parasites released from lysed macrophages can reinvade other cells. A)

SD or LEW BMDMs were infected with GFP-expressing RH (2 hours), washed three times with PBS and

the media was replaced with fresh media containing rabbit anti-SAGI antibody. After 24 hours, cells

were fixed, permeabilized and stained with Alexa Fluor 594 goat anti-rabbit antibody. Parasites are green,

while SAGI is red. The quantification of SAGl-antibody coated parasites was performed with a

minimum of 50 vacuole counts per condition from 3 experiments. (B) Parasites do not shed SAGI upon

invasion of SD BMDMs. Cells were infected with GFP-expressing RH for 18 hours, cells were fixed,

permeabilized and stained with a rabbit anti-SAG primary antibody followed by Alexa Fluor 594 goat

anti-rabbit antibody. SAG I was detected on 100% of parasites in any infected cells. Green = parasite, Red

= SAGI, Blue = Hoechst.

121

6I~

UU

UU

UU

UU

UU

UU

UU

UU

UU

UU

UU

Ua

U NIL>0 LEWVECC3 CDFVECi: LEWLEW0 LEWCDF

SCIDF LEWCID CDF

TOXOPLASMA LETHAL TOXIN

0

Supplementary Figure S7. Overexpression of Nirpi variants confers sensitivity to Toxoplasma and

LT. Viability of LEW and CDF BMDMs nucleofected with full length HA-tagged NLRPI constructs at

36 hours prior to infection with PRU (MOI 1) was measured by MTT assay at 8 hours post-infection.Viability of similarly nucleofected cells was measured 5 hours after treatment with anthrax LT (PA + LF,

each at I pg/ml). Superscripts indicate the NLRPI construct or vector that was transfected into the cell.Graph shows average from three independent nucleofections per condition.

122

100-

50-

0

Mmmummml

5.mlU'

Eupmml

'UEl

'UUK

I.U'

'UU'MUU''U

--- a -- -- - - -

NEW_

J%

A HT1080 lines

100E 76KM RH

50

0

B150 BMAJ lines

0 76K

50-

C Mouse macrophages

100 T

7] 76KF- RH

S50

0

Supplementary Figure S8. Viability of different cell lines and BMDMs overexpressing rat NLRPI

following infection with Toxoplasma. (A) HT1080 fibroblast cells or (B) BMAJ mouse macrophage cell

lines expressing full length HA-tagged NLRPlvara (CDF sequence) or NLRPvanantS (LEW sequence)

were tested for viability following Toxoplasma infection. Infections were with Type I (RH and Type II

(76K) strains (MOI 5) were performed and viability was assessed 24 hours post-infection. Details on

constructions of these lines can be found in (Levinsohn et al. 2012). In select experiments myc-tagged

caspase-l was also transfected 24 hours prior to infection. Values graphed are mean SD, n = 3

wells/treatment. (C) Various mouse macrophage cell lines and BMDMs from mouse strains were tested

for susceptibility to infection as described above. RAW264.7 cells were not tested with the RH strain.

There is no statistical difference between any of the groups or treatments in these studies.

123

Acknowledgments

This research was supported in part by the Intramural Research Program of the National

Institute of Allergy and Infectious Diseases. KMC was supported by National Institutes of Health

(F3 1-AIl 04170), MAH by a Wellcome Trust-MIT postdoctoral fellowship, and JPJS by National

Institutes of Health (RO 1 -AI080621.

124

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128

Chapter Three:Addendum

129

Results and Discussion

Neospora caninum activates the inflammasome in Lewis macrophages

As every strain of Toxoplasma was able to induce pyroptosis in Lewis macrophages

(Chapter Four, Figure 3), we were interesting in determining if related parasite species were

able to activate the rat inflammasome. Neospora caninum is another member of the phylum

Apicomplexa and a close relative of Toxoplasma (Dubey et al. 1988). Neospora and Toxoplasma

share many biological features, including the ability to reproduce both sexually and asexually

and move between definitive and intermediate host. Neospora has a more limited host range than

Toxoplasma and cannot infect humans (McCann et al. 2008). Despite these differences in host

range, Toxoplasma and Neospora have similar genomes with conserved gene content (Reid et al.

2012).

To determine if Neospora was capable of activating the rat inflammasome, we infected

Lewis and Sprague-Dawley (SD) BMDMs. Neospora was able to induce pyroptosis in both

primed and unprimed Lewis BMDMs to the same level as Toxoplasma (Figure 1A). Primed SD

macrophages retained almost 100% viability when infected with Toxoplasma. Interestingly, we

observed a 20% decrease in cell viability in both unprimed and primed SD BMDMs infected

with Neospora.

130

A. - Lewis (-LPS)

100- =I Sprague-Dawley (-LPS)- Lewis (+LPS)[801 Sprague-Dawley (+LPS)

60-

- 40-

20-

B- 6000- M Lewis (-LPS)= Sprague-Dawley (-LPS)- Lewis (+LPS)LE 4000- = Sprague-Dawley (+LPS)

0)

3 2000-

0o1

Figure 1. Neospora caninum is able to activate the inflammasomes in Lewis BMDMs. (A and B) Lewis

and Sprague-Dawley (SD) BMDMs were primed with LPS (IO0ng/ml) for 2-4 hours or left untreated and

then infected with the indicted parasite species (Toxoplasma RH or Neospora NC-I, MOI 1, 18 hours).

Lewis data is average of 2 experiments. SD data is I experiment. (A) Cell viability was measured via

MTS assay. Error bars, SD. (B) IL-I P release measured by ELISA. Error bars, SD.

As expected, host cell death was accompanied by the release IL-I I from primed Lewis

BMDMs infected Neospora and Toxoplasma at comparable levels (Figure IB). Neospora-

infected primed Lewis macrophages released more IL- I P than primed SD macrophages.

However, Neospora infection induced more IL-i P secretion than Toxoplasma infection. This

suggests that Neospora may be able to activate the inflammasome and induce host cell death in

macrophages from both strains of rats to varying degrees.

131

Knockdown of caspase-11 provides protection against Toxoplasma-mediated pyroptosis

Pyroptosis has been linked to two inflammatory caspases, caspase- 1 and caspase- 11.

Caspase- 11 is a member of the noncanonical inflammasome that is activated by Gram-negative

bacteria that reach the host cytosol. Caspase- 11 directly senses cytosolic lipopolysaccharide

(LPS) (Hagar et al. 2013; Kayagaki et al. 2013). Activation of caspase-1 I leads to the cleavage

of gasdermin D (GSDMD), a cytoplasmic protein with no known physiological role (Kayagaki et

al. 2015; Shi et al. 2015). Cleavage is sufficient to initiate pyroptosis. Caspase-1 1 is unable to

cleave pro-IL-I$ (Wang et al. 1996). In some bacterial infections, such as E.coli and Vibrio

cholera, caspase- 11 is involved in the activation of caspase- 1 through the NLRP3 inflammasome

(Kayagaki et al. 2011). The exact mechanism through which caspase-1 1 leads to caspase-1

activation in this model has yet to be elucidated.

We were unable to knockdown Caspi in Lewis macrophages using lentiviral shRNAs

and attempts to block caspase-1 activity using chemical inhibitors did not inhibit Toxoplasma-

induced pyroptosis (Figure 2A). Interestingly, caspase-1 inhibition resulted in a significant

reduction in IL-i IP released from infected macrophages compared to untreated cells (Figure 2B),

suggesting Toxoplasma-mediated host cell death is caspase- 1-independent, while IL-10 secretion

is caspase- 1-dependent.

132

A. B. 10 0 0 -

120 Untreated *M UntreatedWEHD 800. E] WEHD

100. YVAD E YVAD

80, Q-600-

60- - 400S40-

200200

Uninfected Toxoplasma Uninfected Toxoplasma

Figure 3. Chemical inhibition of caspase-1 does not prevent Toxoplasma-induced pyroptosis. (A)

Lewis BMDMs were treated with caspase-l-specific inhibitors Z-WEHD-FMK (WEHD, 50PM) or Z-YVAD-FMK (YVAD, 50pM) for two hours or left untreated and then infected with Toxoplasma (RH,

MOI 1, 18 hours). Cell viability measured via MTS assay. Error bars, +SD. Data using WEHD is average

of five experiments, YVAD is average of two experiments. (B) IL-l P release measured by ELISA. Lewis

BMDMs were primed with LPS (100ng/ml, 2-4 hours) prior to treatment with inhibitors (50pM, 2 hours)

and infection (MOI 1, 18 hours). Error bars, + SD. Data using WEHD is average of five experiments,

YVAD is one experiment. ***p<0.005, paired t-tests.

We were next interested to determine the role, if any, caspase- II plays in Toxoplasma-

induced host cell death. Caspi] gene expression in Lewis BMDMs was knocked down by at

least 6 0 % using three individual shRNAs (Figure 3A). Knockdown was accompanied by an

increase in host cell viability. At least 75% of macrophages from each CaspIi-specific shRNA

condition survived parasite infection, compared to 45% of cells treated with control shRNA

(Figure 3B). This suggests caspase-l I plays a role in parasite-induced pyroptosis in Lewis

macrophages. We previously demonstrated this death was due to the NLRPI inflammasome

(Chapter 3). Although preliminary, this may implicate the canonical NLRPI inflammasome in a

caspase-l I-dependent form of pyroptosis. Thus far, only NLRP3 has been found to be

downstream of caspase-1 I activation.

133

I

A. c 1000UnU 80

X60

40CO,

20

01-

shRNA:0P

4

B. 100

80CU

> 60

040Ca0- 20-

0O01

~ N N *re NN'

SO0 CjP

Figure 3. Knockdown of Caspase-11 protects Lewis BMDMs from Toxoplasma-mediated host celldeath. (A) Knockdown by lentiviral shRNA was confirmed by qPCR. (B) Lewis BMDMs treated withindicted shRNA were infected with Toxoplasma (RH, MOI 0.5, 18 hours) and viability was measured viaMTS assay. Data is 1 experiment. Error bars, SD.

134

CO.1 NSO

I\61)

SO

Materials and Methods

Animals

Lewis (LEW/Crl; LEW) and Sprague Dawley (SD) rats (6-8 weeks old) were purchased

from Charles River Laboratories (Wilmington, MA) and used as source of bone marrow.

Parasites and cells

Human foreskin fibroblasts (HFFs) were grown in DMEM, supplemented with 1% heat

inactivated FBS and 50g/ml each of penicillin and streptomycin and 20pg/ml gentamycin.

Parasites were maintained in vitro by serial passage in HFF monolayers and grown in DMEM,

supplemented with 1% heat inactivated FBS and 50pg/ml each of penicillin and streptomycin.

Toxoplasma gondii tachyzoites from Type I (RH) and Neospora caninum NC-i were

used in experiments. Lewis BMDMs were prepared as previously described (Cirelli et al. 2014).

Cell Viability experiments were performed as previously described (Cirelli et al. 2014).

Reagents

DMEM was obtained from Invitrogen. Antibiotics were purchased from Life

Technologies Corporation. FBS was purchased from PAA. Lipopolysaccharide (LPS) was

purchased from Calbiochem/EMD Biosciences. CellTiter 96 AQueous One Solution Cell

Proliferation Assay was obtained from Promega. Rat IL-i IP DuoSet ELISA, Z-YVAD-FMK and

Z-WEHD-FMK were purchased from R&D Systems.

135

Knockdown experiments

High-titer lentivirus (Broad Institute RNAi consortium) encoding shRNA against murine

Casp4 (also known as Casp]]) was used to infect Lewis BMDMs on day two of differentiation.

The target sequences of the shRNAs are: #1 - GCTCTTGTCATCTCTTTGATA (20/21 bases

match to rat Casp4), #2 - CCGTACACGAAAGGCTCTTAT (20/21 match), #3 -

AGCAACTGAATCTCATTTCTT (19/21 match). Cells were selected with puromycin (6gg/ml).

Knockdown was confirmed by qPCR. Actin primers: 5' GTCGTACCACTGGCATTGTG '3 and

5' CTCTCAGCTGTGGTGGTGAA '3. Casp4/11 primers: 5' TGGACTCAGGCAGCCAC '3

and 5' CTAATGTCAATGTTAGCC '3. Casp4/11 gene expression was normalized against actin

expression levels.

136

References

Cirelli, Kimberly M, Gezahegn Gorfu, Musa A Hassan, Morton Printz, Devorah Crown, StephenH Leppla, Michael E Grigg, Jeroen P J Saeij, and Mahtab Moayeri. 2014. "InflammasomeSensor NLRP1 Controls Rat Macrophage Susceptibility to Toxoplasma Gondii." PLoSPathogens 10 (3).

Dubey, J P, J L Carpenter, C A Speer, M J Topper, and A Uggla. 1988. "Newly RecognizedFatal Protozoan Disease of Dogs." Journal of the American Veterinary Medical Association192 (9): 1269-85.

Hagar, Jon A, Daniel A Powell, Youssef Aachoui, Robert K Ernst, and Edward A Miao. 2013."Cytoplasmic LPS Activates Caspase- 11: Implications in TLR4-Independent EndotoxicShock." Science 341 (6151): 1250-53.

Kayagaki, Nobuhiko, Soren Warming, Mohamed Lamkanfi, Lieselotte Vande Walle, SalinaLouie, Jennifer Dong, Kim Newton, et al. 2011. "Non-Canonical Inflammasome ActivationTargets Caspase-1 1." Nature 479 (7371): 117-21.

Kayagaki, Nobuhiko, Michael T Wong, Irma B Stowe, Sree Ranjani Ramani, Lino C Gonzalez,Sachiko Akashi-Takamura, Kensuke Miyake, et al. 2013. "Noncanonical InflammasomeActivation by Intracellular LPS Independent of TLR4." Science 341 (6151): 1246-49.

Kayagaki, Nobuhiko, Irma B Stowe, Bettina L Lee, Karen O'Rourke, Keith Anderson, SorenWarming, Trinna Cuellar, et al. 2015. "Caspase- I1 Cleaves Gasdermin D for Non-Canonical Inflammasome Signaling." Nature advance on (September).

McCann, Catherine M., Andrew J. Vyse, Roland L Salmon, Daniel Thomas, Diana J.L.Williams, John W. McGarry, Richard Pebody, and Alexander J. Trees. 2008. "Lack ofSerologic Evidence of Neospora Caninum in Humans, England." Emerging InfectiousDiseases 14 (6): 978-80.

Shi, Jianjin, Yue Zhao, Kun Wang, Xuyan Shi, Yue Wang, Huanwei Huang, Yinghua Zhuang,Tao Cai, Fengchao Wang, and Feng Shao. 2015. "Cleavage of GSDMD by InflammatoryCaspases Determines Pyroptotic Cell Death." Nature advance on (September).

Wang, S., Miura, M., Jung, Y. k, Zhu, H., Gagliardini, V., Shi, L., ... Yuan, J. (1996).Identification and characterization of Ich-3, a member of the interleukin-I beta convertingenzyme (ICE)/Ced-3 family and an upstream regulator of ICE. The Journal of BiologicalChemistry 271(34): 20580-7.

137

Chapter Four:Three novel Toxoplasma gondii dense granule proteins are required

for Lewis rat NLRP1 activation

Kimberly M. Cirelli, Vincent Butty, Musa A. Hassan, Jeroen P.J. Saeij

Kimberly M. Cirelli contributed to Figures 1, 2, 3, Si, S2, S3. Vincent Butty and Musa Hassancontributed to Figures 2A.

138

Abstract

Toxoplasma gondii is an intracellular parasite that can form a lifelong chronic infection in

hosts, characterized by cysts in muscle tissues and the brain. The Lewis rat is unique in its

ability to completely clear Toxoplasma infection and prevent the establishment of a chronic

infection. Previous findings established that Lewis macrophages undergo rapid cell death, known

as pyroptosis, when infected with Toxoplasma. Pyroptosis was controlled by the NLRP1

inflammasome. We have used a chemical mutagenesis screen to identify Toxoplasma genes

involved in NLRPI inflammasome activation. We isolated several parasite mutants that induce

significantly less pyroptosis in Lewis macrophages and reduced IL- 1P secretion. Utilizing whole

genome sequencing of our mutants, we identified single nucleotide polymorphisms and

deletions. We then used CRISPR/Cas9 to knockout individual candidate genes. We have

identified novel Toxoplasma dense granule proteins, GRA18, GRA27 and GRA28, which are

individually required for inflammasome activation in vitro. Strains deficient in GRA 18, GRA27

or GRA28 display the same phenotype as the mutants isolated from the screen. Complementation

of mutants with wild-type allele of Gra18, Gra27 or Gra28 is sufficient to restore ability to

induce pyroptosis in Lewis macrophages.

139

Introduction

Toxoplasma gondii is an obligate intracellular parasite that infects all warm-blooded

animals (Hill and Dubey 2002). Among its different hosts, there are natural differences in

susceptibility to the parasite. Rats and humans are relatively resistant to Toxoplasma. Most

members of both species are asymptomatic upon infection, but the parasite establishes a chronic

lifelong infection by developing into cysts in brain and muscle tissues. However, the Lewis rat

strain, can clear the parasite and fails to develop this chronic infection (Sergent et al. 2005). This

resistance was mapped to a single locus, Toxol (Cavailles et al. 2006) and correlated with

induction of pyroptosis by Toxoplasma in vitro. Toxoplasma-induced cell death in Lewis

macrophages was determined to be controlled by NlrpJ, which encodes for the NLRP1

inflammasome sensor (Cirelli et al. 2014; Gorfu et al. 2014).

The inflammasomes are a family of cytosolic pattern recognition receptors (PRRs).

Activation of the sensor, leads to the formation of a multimeric complex and the recruitment and

proteolytic activation of pro-caspase-1. Caspase-1 cleaves pro-IL-1 and pro-IL-18, resulting in

their release from the cells. Caspase-1 activation can be accompanied by host cell death, termed

pyroptosis (Lamkanfi and Dixit 2012; Kayagaki et al. 2015; Shi et al. 2015). Pyroptosis has been

established as a host mechanism to clear intracellular pathogens, particularly Salmonella

typhimurium and Legionella pneumophila (Miao et al. 2010; Miao et al. 2011). NLRPI

activation in Lewis bone marrow-derived macrophages (BMDMs) results in the release of IL- 18

and rapid death of the host cell, releasing Toxoplasma into the extracellular space before parasite

replication can occur. In conditions where pro-IL-1p expression is induced in macrophages (e.g.

LPS-primed), infected cells also release bioactive IL-i$ (Cirelli et al. 2014). As macrophages

are among the predominant cell type infected upon an oral infection, it's likely that macrophage

140

pyroptosis is a host mechanism to prevent parasite proliferation and dissemination (Mordue and

Sibley 2003; Lambert and Barragan 2010).

The specific stimuli that can activate the inflammasomes and their mechanism of

activation vary. NLR family CARD domain-containing protein 4 (NLRC4) recognizes NLR

family, apoptosis inhibitory protein (NAIP) proteins bound to bacterial components, namely

flagellin and type III secretory system proteins (Kofoed and Vance 2011; Zhao et al. 2011).

Anthrax Lethal Toxin (LT) is a protease and a direct activator of rat NLRP1 (Newman et al.

2010). LT cleaves the N-terminus of NLRPI in LT-susceptible rat macrophages. This cleavage is

sufficient to activate the inflammasome and induce pyroptosis (Levinsohn et al. 2012). Because

the types of ligands and modes of activation of the inflammasomes are varied, we chose to take

an unbiased approach to identify the Toxoplasma gene product(s) required for Lewis NLRP1

inflammasome activation. Using a mutagenesis screen followed by whole genome sequencing

we identified three novel Toxoplasma dense granule proteins (GRAs) required for NLRP1

inflammasome activation in Lewis rat macrophages. Parasite strains deficient in individual

proteins induce significantly less pyroptosis and IL-i IP processing in vitro.

141

Results

A mutagenesis screen isolates parasites that do not activate the NLRP1 inflammasome

We previously found that upon infection of Lewis bone marrow-derived macrophages

(BMDMs) by Toxoplasma the NLRP1 inflammasome is activated leading to rapid host cell death

and the control of Toxoplasma replication (Cirelli et al. 2014). To identify Toxoplasma gene(s)

required for NLRP1 inflammasome activation, we designed a chemical mutagenesis screen to

enrich for parasites that fail to induce pyroptosis in Lewis BMDMs (Figure 1A). Five

independent populations of chemically mutagenized type I (RH) parasites were used to infect

unprimed Lewis BMDMs for two hours. Extracellular parasites were washed from cells and

media was replaced with fresh media that contained the glycosaminoglycan, dextran sulfate.

Dextran sulfate acts as a glycan competitor and prevents host cell invasion by extracellular

parasites (Carruthers et al. 2000). Parasites that retain the ability to activate the NLRP1

inflammasome are released from the lysed cell into the supernatant, where the parasite is coated

with dextran sulfate, blocking invasion into a new host cell. Mutated parasites unable to induce

pyroptosis are able to replicate within the surviving macrophage. After 24 hours of infection,

surviving cells were washed, thereby removing the extracellular parasites capable of inducing

pyroptosis from the population. The parasites within the macrophages were then allowed to

continue replication until their natural egress from the macrophages.

After five to nine rounds of selection, single parasites were cloned from the populations

and individual clones were tested for their inability to induce pyroptosis. Eleven mutants were

isolated and determined to induce significantly less pyroptosis in Lewis BMDMs. By sequence

analysis, we determined several clones were identical. Overall, seven unique mutant strains were

142

isolated. We chose to focus on four independent clones (#1-4). At least 75% of Lewis

macrophages infected with any mutant strain survived, which is in contrast to only 25% of

BMDMs that survived infection with the wild-type strain (Figure 1B). As expected, survival of

the host cell was linked with the ability of the parasite to replicate within the macrophage. After

24 hours of infection, 80% of the surviving macrophages infected with wild-type parasites

contained only single parasites compared to those cells infected with each mutant strain, in

which only 25% of infected cells contained single parasites (Figure 1C).

143

A.

Mutagenize parasites

Infect BMDMs for 2 hrsChange media ( + DS)

Parasites activateinflammasome and

are released intosupernatant

Failure to inducepyroptosis allows

for parasitereplication

E.D.

150kD-

37kD-

*

)

Ct

C

100.

80.

60-

40-

20-

0.

B

CU

. ,, **

**

100

60

40

2

0- r

C. 100 WT

T 80 W Mutant #1a Mutant #2

60 W Mutant #3a a) Mutant #4

c 40.

20

01 2 4 8

parasites/vacuole

4*4

IIV

17kD- 4W

Figure 1. Isolation of Toxoplasma parasites that do not induce pyroptosis. (A) Schematic ofmutagenesis screen. DS is Dextran Sulfate. (B) Lewis BMDMs were infected with indicated strains(MOI= 1, 24 hours). Macrophage viability was measured via 3-(4,5-diiethylthiazol-3-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Data shown is average of 7experiments. Error bars, + SD. **p<0.005, ****p< 0 .0 0 0 1, paired t-test (C) Number of parasites pervacuole in infected BMDMs (MOI = 0.5, 24 hours) as measured by microscopy. Between 50-100vacuoles counted per experiment. Average values from 3 experiments. Error bars, +SD. P-values are<0.0001, two-way ANOVA comparing mutants to wild-type. (D) Western blot probing for IL-lp onconcentrated (20X) supernatants on LPS-primed (100ng/mI, 2 hours) infected with indicated strains (MOI= 1, 24 hours). Image is representative of 2 experiments, pro-IL-l is 37kD, active IL-1 , aspecific bandis represented by asterisk and indicates similar loading of samples. Mutant 4 was not tested. (E) LewisBMDMs were primed with LPS (100ng/ml, 2-4 hours) and infected with indicated strains (MOI =1, 24hours). Viability measured by MTS assay. Data shown is the average of 4 experiments. Mutant 4 was nottested. Error bar, + SD. ***p<0.0005, paired t-test.

144

I

MILL-- z-1002

10

TT

Inflammasome activation is marked by cleavage and secretion of the pro-inflammatory

cytokines, IL-1p and IL-18. To induce expression of pro-IL-i IP, BMDMs were primed with LPS

prior to infection with Toxoplasma. To measure active IL-i IP, we subjected the supernatants of

infected BMDMs to Western blotting, probing for both the inactive (37kD) and bioactive (l7kD)

forms of IL-i I3. We found a strong decrease in the amount of cleaved, active IL-i IP secreted from

macrophages infected with each of the mutant strains, compared to wild-type (Figure 1D).

Significant amounts of the inactive form of the cytokine were also measured in the supernatants.

We noted that LPS-primed macrophages infected with mutant parasites survived less than

unprimed macrophages, but still significantly more so than wild-type infected BMDMs (Figure

1E). This increase in host cell death of primed cells most likely explains the large fraction of

inactive IL-i I released from the cell.

Toxoplasma also activates the inflammasomes in mouse macrophages. Several reports

have implicated both NLRP3 and/or NLRP I activation in the murine response to control parasite

replication. Although the role of pyroptosis, if any, in this resistance is not fully understood

(Gorfu et al. 2014; Ewald, Chavarria-Smith, and Boothroyd 2014). Unprimed or LPS-primed

C57B1/6 macrophages showed no significant difference in host cell survival after infection with

wild-type or any mutant strain (Figure 2A). Mouse macrophages infected with mutant strains

secreted more IL-lp than wild-type infected cells (Figure 2B). These results suggest that the

inability of the mutants to activate NLRPI inflammasomes is a rat-specific phenomenon.

145

A. Unprimed120 LIII Primed w/ LPS

. 100- 80

> 60040-

200 * - - - - - -

B.125

100- - Unprimed

C~W Primed w/ LPS7&-

50

25j

0*

~<N

Figure 2. Mutants activate the inflammasome in mouse BMDMs. (A and B) C57BL/6 BMDMs were

primed with LPS (100ng/ml, 3 hours) or left unprimed and infected with indicated strains (MOI 1, 24

hours). (A) Cell viability as measured via MTS assay. (B) IL-1I3 secretion as measured by ELISA. Error

bars, + SD. Data are from I experiment.

146

Identification of mutated genes

To identify the genes mutated in each clone, we performed Illumina whole genome

sequencing on each strain. Each clone had at least five non-synonymous mutations (Table 1).

Two clones (mutant #3 and #4) shared one mutated gene, TgGTJ_226380 (Figure 3A).

Mutations in both clones resulted in a premature stop codon. Mutants #1 and #2 did not have any

genes mutated in common with any of the other isolated strains. To identify the causative

mutations in mutants #1 and #2, we established a set of criteria to narrow the list of possible

genes. The inflammasomes are expressed within the cytoplasm of host cells. We hypothesized

that a Toxoplasma secreted protein that can interact with host cytosolic proteins might be

recognized by NLRP1 or modulate the activity of the inflammasome. We therefore chose to

focus on genes whose protein products contained predicted signal peptides. Additionally, we

previously tested numerous strains of Toxoplasma that are genetically distinct from the clonal

types I, 1I and III for their ability to activate the inflammasome. All strains tested were able to

induce pyroptosis (Chapter 3, Figure 3). We therefore focused on genes that were expressed

(FPKM>10) across all strains that we tested using a previously published RNAseq dataset

(Minot al. 2012). Using these criteria, we narrowed the list of candidate genes in mutants #1 and

#2 to three genes each (Figure 2A).

147

Chromosome Position Ref Sub Codon AA Gene MutChange Change

TGGT1 chrXII 3698939 C T Cgt/Tgt R/C TGGT1_248260 1TGGT1 chrXI 4323464 A G cTc/cCc LIP TGGTI_314875 1TGGT1 chrX 5454719 A T Tga/Aga */R TGGTI_236870 1

TGGT1 chrVIII 3546892 A G Aca/Gca T/A TGGT1_273510 1TGGT1_chrVIlb 258249 C G Ccg/Gcg P/A TGGT1_263360 1TGGT1 chrVIlb 1300287 A G tTc/tCc F/S TGGT1_262825 1TGGT1 chrVIlb 4053654 G C Ccg/Gcg P/A TGGT1_257500 1TGGT1_chrVIla 683027 A G Ttc/Ctc F/L TGGT1_206550 1TGGT1 chrVIla 1666878 A G tTg/tCg L/S TGGTI 204310 1

TGGT1 chrV 2683109 A C Ttg/Gtg L/V TGGT1_284040 1TGGTIchrIX 1745808 G A cCc/cTc P/L TGGTI_264890 1TGGT1 chrIX 3803976 T C Tct/Cct S/P TGGTI_290960 1TGGT1_chrIll 527809 A T aaA/aaT K/N TGGTI_252395 1TGGT1_chrIll 1241431 C T Gac/Aac D/N TGGT1_253870 1TGGT1 chrlb 814454 A T cTg/cAg L/Q TGGT1_208580 1

TGGT1_chrVIla 2153702 GGA GA gag/aga E/R TGGTI_204050 1TGGT1_chrVIla 2964132 C G ttG/ttC L/F TGGTI_203040 2TGGT1_chrVI 290424 T C Aaa/Gaa K/E TGGT1_239130 2TGGTI chrVI 3356628 G C Gga/Cga G/R TGGTI_243635 2TGGT1_chrV 121175 A G aTc/aCc I/T TGGT1_220175 2

TGGTIchrXII 5959927 A C cAt/cCi H/P TGGT1_278518 2TGGT1 chrVIII 2061823 T C Agt/Ggt S/G TGGT1_231410 2TGGT1_chrVIlb 730342 C T Cgt/Tgt R/C TGGT1 264140 2TGGT1_chrVIlb 2573674 G A cCa/cTa P/L TGGTI_260450 2TGGT1 chrVIlb 3451345 A G gAt/gGt D/G TGGT1 258580 2

TGGT1_chrX 5567109 C T tGg/tAg W/* TGGTI_237015 2TGGT1_chrIX 2023395 T C gAa/gGa E/G TGGTi_264472 3TGGT1_chrV 1043175 T C Acg/Gcg T/A TGGT1_213610 3TGGT1_chrVI 1514625 A T gAt/gTt D/V TGGTI_240960 3TGGT1 chrVI 674303 C T aGa/aAa R/K TGGTI_239700 3

TGGT1_chrVIla 1197377 C G Gcc/Ccc A/P TGGTI_205160 3TGGT1_chrVIII 2566377 G T gaG/gaT E/D TGGTI_233120 3

TGGT1_chrX 1583637 A T Aaa/Taa K/* TGGTI_226380 3TGGT1_chrX 3027043 T A Agc/Tgc S/C TGGT1 224280 3TGGT1_chrX 401396 T C Agt/Ggt S/G TGGTI_228210 3TGGT1 chrXI 2517037 A G cAc/cGc H/R TGGT1 312140 3TGGT1 chrXII 1102624 T C Aag/Gag K/E TGGT1 219070 3TGGT1 chrXII 6691803 T C aAg/aGg K/R TGGT1_277030 3TGGT1_chrIll 1572757 T C Tca/Cca S/P TGGT1_254300 4TGGTIchrIV 666512 T C gAa/gGa E/G TGGT1 319590 4TGGT1 chrV 2817686 A T caT/caA H/Q TGGT 283780 4TGGT1_chrV 1208069 A G gAa/gGa E/G TGGTI 213790 4

148

TGGTI_chrVlla 3738267 T A gTt/gAt V/D TGGT1_202120 4TGGT1_chrVIla 2285765 T A aTg/aAg M/K TGGT_1 202210 4TGGT1 chrVIlb 1784927 A G Tac/Cac Y/H TGGT1 260800 4TGGT1_chrVIlb 174292 A G gTg/gCg V/A TGGT1_263230 4TGGT1 chrVIlb 4144483 T A Tcg/Acg S/T TGGT1 257670 4TGGT1 chrVIII 1082227 C G Ccc/Gcc P/A TGGTI_229750 4

TGGTIchrX 1583588 T A taT/taA Y/* TGGT1_226380 4TGGT1_chrXI 1897511 T G tTc/tGc F/C TGGTI_313050 4TGGT1 chrXI 5986311 A G gTc/gCc V/A TGGT1_216590 4TGGT1 chrXII 6017381 T A Tcg/Acg S/T TGGTI_278440 4TGGTI chrXII 3854449 T G Aaa/Caa K/Q TGGTI _248510 4TGGT1_chrXII 5321855 T C Tct/Cct S/P TGGT1 251450 4TGGT1_chrXII 6356740 T A Tcg/Acg S/T TGGTI_277895 4TGGT1 chrXII 331776 G A aCg/aTg T/M TGGTI _307760 4TGGT1 chrXII 586725 G C tCt/tGt S/C TGGT1_219790B 4TGGT1 chrXII 586722 G T tCt/tAt S/Y TGGT1_219790B 4TGGT1 chrV 2177685 C T cGa/cAa R/Q TGGT1 285650 4TGGT1 chrlb 322251 A T Tgc/Agc C/S TGGTI_207800 5

TGGT1 chrVIII 1119911 T C Tcc/Ccc S/P TGGTI 229690 5TGGT1_chrXI 207115 T C aAt/aGt N/S TGGT1_308810A 5TGGT1_chrXI 669869 G T Gag/Tag E/* TGGT1_309160 5

asmbl.726 5415 C G Ccc/Gcc P/A TGGT1 323020 5asmbl.1029 1675 T A Tat/taA Y/* TGGT_1 411420 6asmbl.1059 7 G T Gca/Tca A/S TGGT1__256220 6

TGGT1_chrlb 1566573 C A aCg/aAg T/K TGGT1_209920B 6TGGT1 chrVIla 3144810 T C gAc/gGc D/G TGGTI_203230 6TGGT1_chrVIII 5737106 GA TT tttcca/ttAAca FP/LT TGGT1_269885B 6

TGGT1_chrX 6716961 G T aaG/aaT K/N TGGT1_215220 6TGGTI chrX 4066658 T A Aac/Tac N/Y TGGT1_234165 6TGGT1 chrXI 3304365 A G aAc/aGc N/S TGGT -_310910 6TGGT1 chrXII 557779 A T tTt/tAt F/Y TGGT1 219820 6TGGT1_chrlb 1446051 C G cCa/cGa P/R TGGT1_209755B 7TGGT1_chrlb 205995 A T aaA/aaT K/N TGGTI_207650 7TGGT1 chrIll 634457 T A aAg/aTg K/M TGGT1_252880 7TGGT1 chrIX 4831825 T A Tgg/Agg W/R TGGT1_410730 7TGGTI chrIX 5429427 G T Gaa/Taa E/* TGGTI_306338B 7TGGT1 chrV 1801863 G T aaG/aaT K/N TGGTI_286160B 7

TGGT1_chrVIlb 2849542 T C atA/atG I/M TGGT -_259290 7TGGT1 chrXI 5534773 A T caA/caT Q/H TGGT1 316780 7TGGT1_ chrXII 6219164 A T Act/Tct T/S TGGT1__408760 7TGGT1_chrXII 259389 G T Gtg/Ttg V/L TGGTI 410880 7

Table 1. List of all identified non-synonymous mutations. "Ref' is reference nucleotide(s) in wild-typestrain (GT1 v9.0). "Sub" is nucleotide variant(s). "Mut" is mutant clone number.

149

We individually disrupted each candidate gene in RH using CRISPR/Cas-9 and tested the

resulting strains for their inability to induce pyroptosis (Sidik et al. 2014). Knockout of most

genes resulted in no difference in inflammasome activation compared to wild-type (Figure 3B).

Parasites that contained single disruption of three different genes, TgGTJ_226380,

TgGTJ_237015 and TgGTJ 236870, induced significantly less host cell death upon infection

compared to wild-type parasites. Complementation of the knockouts with the disrupted gene

restored their ability to induce host cell death (Figure 3C).

We have previously shown that multiple strains of Toxoplasma and Neospora caninum

are capable of inducing pyroptosis in Lewis macrophages (Chapter 3, Figure 3 and Chapter 3

Addendum, Figure 1). We saw similar results when these genes were disrupted in type II

(ME49) parasites. Lewis macrophages infected with parasites deficient in each of these genes

retained 100% of cellular viability compared to 20% of wild-type infected cells. (Figure 3D).

Homologs of TgGTi_226380, TgGTJ_237015 and TgGTJ_236870 were identified only

in two members of the Sarcocystidae family, Neospora caninum and Hammondia hammondi

(Figure 4). The predicted protein products of these genes lack predicted functional domains. The

resulting proteins each have two predicted transmembrane domains and several alpha helices

(Figure 5).

150

A. Gene ID MutationMutant #1

TgGT1_236870 X214R(GRA28)

TgGT1_248260 R10CTgGT1_204050 R57fsX72

(Sub1)Mutant #2

TgGT1_237015 W175X(GRA27)

TgGT1 203040 L623F

TgGT1_258580 D138G(ROP17) D18Mutant #3 & 4

TgGT1_226380 K138X(GRA18) Y121X

B.

100-

>80-

60-

4020

0 QP0eNo( O $

D.

150-

100-

0 50-

0

C.

100-> 80-

60-

-0>40-

20

-10 A& ..b\ O

0\c (

4 79

Figure 3. Three genes are individually required to induce pyroptosis in BMDMs. (A) List of genes

containing non-synonymous polymorphisms that fulfill candidate gene criteria in isolated mutants. (B)

Toxoplasma parasites were individually knocked out for candidate genes using CRISPR/Cas-9, with the

exception of Subl. Lewis BMDMs were infected with indicted strains (MOI =1, 24 hours) and cell

viability was measured using MTS assay. Two clones deficient in each individual gene was tested at least

twice. Graph is average of one clone per gene. Error bars, + SD. (C and D) Cell viability as assessed by

MTS assay of Lewis BMDMs infected with indicated strains (MOI = 1, 24 hours). Each strain has been

tested in at least three experiments, with the exception of ME49AGRA28, which has been tested once.

Error bars, + SD. ***p<0.0005, ****p<0.0001. paired t-test.

151

A.AA..A~.-..Adfl23.A5A 7875 105

Tq T1 226380 -MY PLTVY .I"TgVEG_226381) M YPLTV YW ITgKE49__226 38 - YPLTVYS IHMA_226380 HYPVTV" INCLIV_04 6581) 11:LI Y TP INCLTV_047520 1*QLPI - , V ,k 6nttns 87-641- 39

TgGTI_226380 AS PVNRPR PRTgVEG_226380 AS PVNR PiPK

TgME69_226380 AS PV MR P IPCI HA_226380 ASPIVNRRTPKNCLIV_04658 BUSSAVDL.RMSK

NCLW_04 75 20 VSERNIAX A(~nhec I - 51 8S 4S4' I

TgGT1_226380 PRPRLRKSQR RSRSLBRRQR ITgVEG_22630 PRP RL KTQR RSRPLERRQi 1

TqA49_226380PRPRLR SQR RSSL S RAQR 1RHA_226380 PRPRFRK SQR RANTL 5RRQR IMCLIV__4658" -LRYRKPRV x-TSN -- RRRQ I

HCimV_04752t) LVSHTAVQKS RPGGa ROWLR ICos en 41157 477 57 5 85 04457 7 660 a

R YGEA91V01 TQRRGLARVT.TGRASV.1' TQRRGLARVTRYGE ASVD:: TQRIIGIARVTRORRAYVDD - TRRGLARVT

-- LHVDYEDA TP PRGLAR RT- -DDLVHRG -VKKDDTAAPR

4 2 3a6 45 8 70 8 56 77 7 * 7 5 7

"-VVGAAIA:' KTWYAKRtQ- -"-V VGAA IAF KT YAK It-QA--VVGAAIAV* KTWY KitQA-VVGAkVAL KAWY RR Q-A- IVGAA.1V XAAYSRRQ -

STLLGAYAF RKARK IR I X*&7~~~~~ 86'6 57a 09

EARDLPL AMLYE LKEA NRAVVTVD DQLZRLNRQL P IVMDPDYADRDLPL SLYQQLAK E A ARRRVVTVD DQLERLNRQL P:: A UDA

RR D.L PAL .LYQQLAQA ICAITSTVD DQLERL IRQL PMPVGMDPDVEAllDR DLPSL" STLYHQLQR A KSQRVVTTLN DQL GRLNRUL P liI VQMPDY* RRR&DLPTL J S LYQQMEEA I RQ VT STLD EQITALMR QV Per.V OVD PDYMAERRSSPT. NLL ADQLVE:A 11KGRTA KRL SYG VSDOL PSTVOM -- T

56 a8 8 7 6 * * 5?* 6 86 777' 1 7 784687 -oe _5 . .96 * 66 7

TgGT1__2263B0 YKLRERALFCT TRGRADVAQA LVDGLLTERM KVNILINRAR Q-t';-IAKGTA

TgVEG_-226360 YIILRERAVCX TII:ADVAQA LV.GLLT:Re VIIL ...A. QEIA..TATg AA49_226380ALRERACD TRO ADVAQA LVDGLLT I KVNIL114 A 14 Q- SDAKGTA

HAAA_226380 A ALLARAFCY TRGADVAQA LVGGLLTe KVNVLIRAR N PELAAGTALMCLIV_046S0 NKLRAHArx XYGAGVAER AAL.LEEAQRR LVRLLGVR VAVRAAVTD

ACLIV 047S20 ITAAA I LR AAEAAGR AS'S??KSD AADES15AR NAMAVATPACons~ng 557 66 76 67 ** 8 66 7 737 -5676 6868 5 8 4646 5 6*5

AXEEU LYTIIL PLRG--PAYVRAM EALYP T LRG .AS RA EE LHTPL PPRA PA IV I

A QaLLEFT. PA 1PV'DNIASASE GAS-P'aVV

*067P775656 7395%*6. 7

MSEDVLRQ~v AEk'KQT--MSEDVLRQVL) AEPKQT--

M:EDVLRtQVQ AEPKQT --

K QEAL:QVV VD-RGT -

HAADV14 QAM QVKTGT --

L A KIC QL TPPV:D=9 5 69*76 5 47 5 7

B.TgGT1_237015 X-TVPVHLTGTgVEG 237015 A-TVPVHLTO

TgNE49_237015 X-TVPVHLTGTqLA_237015 N-AVPDDVGNCBN1204_05091 XVVPAKLLL

Cassistk' . 65585-

PRENLEA YVAETAATVQ ESTRRPPRWR DAPEPTSRAl GTEEEDAD TA

rl.R:ELEA YVAETIAATVQ EWTRRPFRWR GAPEPTSRAM GTEEEDADT APFRSRENLEA YVAETAATVQ EWTARPPLA DAPEAT RAS GTEESDADTD

YPRE ITLTD YVAETVSAVD WT RRLQPR DVPPTPRAE GTEEEEAEIA

YRP5VYLTD DLYASRGVW QWARG-- AT LADAGGSAA AVT7AQLRAA86 .S 65*-55 68 67665-4 a -7-.34 37 487766S7*.7 77 7776755-

?gGTA_237015 C---SAADm DVAV5DGGHE SAGLVSDIAQ GHKTRGLD

TgVEG_237015 C---SAANDM DVAVDGGHE SAGLVEDIAQ GHKTRGLDTgRE49_237015 C--- SAADH DVAVADGGHE AGLVEDIAQ GHKTRSGLDE

HHA_237015 C-- CMASDT EVAVDEHD SAGLVBDIAP GHKTRSDLDE

MCN1204_ 5091C&0DVAIGM QSAASV-GDV SAELIDTPG GKEQPSDA

( OC001en5 6 ' 56 7 7 67-86-63-1 5 - - 6-9* 77- '-* "*10-4678

LSCrLVLTGHLSCrLVLTGH

LSCFLVLTOHLYYPLVLTGO

LYCFSVSTGH.56. 7 .7 *

STSTTSRQRRSTSTTSRQR

ISTSTTSRQ.RRSASTTSRQRRNISTSRRPRE

AVLAXCVALR

AVLAICVALR

AVLAICVArR

AVLAIFVAFRAALAMCLAYR

ARSRLREQAQ D---HVRSVR TGLD-A-RB

ARRLREQAQ D--- VRTR TGLD-A-RV

ARRLREQAQ D---HORP R TGD-A-RV

ARARFREQVQ a_---DHRAVR TGLE-AEAD

-ASAVLAVVAD D LAAQVQ PELTIR LGD7 767766 7 80657kA68 76 6 7$6-3

TgGTI_237015

TgVEG_237015TqHE49_237015HHA_2370155

WCBN1204_ 0509,

QAKYGRVRFSQAKYGRVRrSQAKYGRVRTS

C.TqGT1 236870 - A

TqVEG 236870 -- AAATgqA49 236870W--AR?

mA 236870 -.- A

NCLV 050780PConsisko7 7 6

VASWQSSERPVHSWQSSKRP

VmAwQ ERP

LH WQ "IRRLM SWH A? 0R?

FL PPVRRQCPLPPARROQ

FPWVPVRRQLLAIP ---- - R"

14*66",8 R

DRPPEYTDGG APOGG

DRPPEYTDOG AIG.6G. PEYTDGG APOS

DQP PPYTRG PPGSGHLPPRYEA-- PPDQG75--*5 -7436 5-77 *

LPRLRPIFLELP_'RLRPVLH

LA 7'LRPFLHLP-TLRPFVL'-LP: K6 IA-'** I-i

SLIA QVGVAA

A LIAOVGVAA

A LAPOVGVAA

SLIFQVGVAA

SVIS; LGVAA

;iTKEAGURVERTKKAGW RVE X

RA KAGCN RVK IST G GHAM UD

ILFSOMIPGGILV:DMIPGGLVSD:IPGG

ILL D IPGADLLLDTPPGL

L L67P 7P 7.

LTVGVLAAAL AORAAPPOL PA-PVP-AARLTVAVLALLL AQRAPPQL PA-FVP-ALRLAVAVLALIL DQRAATPPQL P--FVP-ALRLrVTILAIL WQQR-'APLRM PA VRE ALT

VAVTILGL L YRRSXAEG PA6EEP ULT8A659* ."S. 7884557 7? *t4*73 6

P -AS 3ALLGSPH -TPG--2DXL GAURRT:D:L

PAS SALLGDSPH &TPGREED L GADRRTASL

PPAS SAMLLGDSPA DTPGAAEDA L GAHARTUDAL

GPA| RAMLLGDPPAY VTAGUEERA GAARTSDL

LPVP PPATL'DSPA UTPAGGNR OTDGAASYG

3A87 5777-.N5 6'5. 6676 *7S6776E6

POQQLIPP-Y PWTAAANP N---------PADQLPPP Y MPPARVAARP N-

PGQQLAPP-Y SPTSVATNP A

P0--A-PP-Y EPMRRVASAP S

YA---PPY EPURRVARRP 4,0

6 .3363'.'O . - 79D5 7

EDAVAVPVD KILAAHYtLP LDAVTAYPEDESVSPVD ZRLAAYRLP LDAVDTRRYP

AASVSVPVD 9LSAKTItLAP LDAVDTRAYPEDESVSVPVD I R,:AR91;YRAPVP LD)AVDTRRYQ

QPVIPV0 KRVAAPTAFP LREVDTRRSP

7 _ _ 77,_ 7

Figure 4. TgGTI_226380, TgGTI_237015 and TgGTI_236870 have homologs in Neospora andHammondia. Alignments of primary peptide sequences using Profile ALIgNmEnt (PRALINE), scoringamino acid conservation. (A) Alignment of Toxoplasma gondii TiGT 226380, TgME49 226380 andTgVEG 226380, Hammondia hammondi HHA -226380, Neospora caninun NCLIV 046580 andNCLIV 047520. (B) Alignment of Toxoplasma TgGT1 237015, TgME49 237015 and TgVEG 237015,

Hammondia HHA 237015 and Neospora predicted protein BN1204_050915. (C) Alignment ofToxoplasma TgGTI_236870, TgAIE49 236870 and T:g VEG 236870, Hammondia HH.A 236870 andNeospora NCLIV 050780. The legend above depicts color scheme. Mutations are marked by red asterisksabove mutated base.

152

I

ATMRGS AW-

SETMRDSAV-ACAMROSGR-

EETVNRSHTI~1- 7 o7 4- 54Wil

TgGT 236870TAVEG 236870

TqmE49 2367TA 23670

PICLIV 01078C

TgGTi 236870TAvzG 23687A

TgAD49_23687

FA 236870SCLAv 000780C ARDIAy A

LRNLGV

LPRMLGV-

LRNLGFPSNLSPSSELGF467 .7 .

PVQQTETRAE

PF QQTETRAPFQQTETRASPOTUTRAiTTRRnTG^s76 877 6**

DGKVLRPVRADGRVLSPVBA

CDGRVLPVHA

DGAVQPLRA

TRRSQXTRUT76975*7577

U XeEWLDDD0SKEELDDr

DSKEEULDDU

DWNREEPDDD

DSORRXYDDEA6 ADLA* -

LAIV.GLLVCZ"LA VVGL'.LVC

LAVVGL-YCLAVVGLNLVC

L.AAVAXIGrVC

VOVVIAMH -sea*5765!6

AAAKSNSEE T

AAAKSTSEE TAAA-SNSEXTAAAF.N.HGTADAE .. .I

:57653543 7

EHRELVKVRE,ZHR:LVKVR

EH R.LVKV:E

:HR.L'VVRIIIBL - TRQ

.7777677.8 1

1VRT1PDKAQ J

, VRTDPDKAQ J'L.VRITD PDK AV II VRITEPDK AK IN 0GTNA - ALQ AYPL.T 9 E'E A. J

645S. l5 ' I .

it WQQLQPE

K.PWQQLQP-KHF.QQLQPISKHFWQQLQP

R RPWNRLHP RI DFL IRFLA I15*1 67 o S7 6

VFCPERSRRS

VFCP::SRRSVTCPRSIRRSVFCPXR SAR 5IPCPERPRR:96e-- -7**

. I R. ....L4NRHDRPTVHLURHDRPIVHL

" RHDRLTH ILDRTSCATLRV

0*6 7 6 7768

PHVPRLPPT Y ZPMVPRLPPTY z

PM4VPRLPPTY Z

LVI:REPPTY A

PI GP, --- Y I65*631'-

'4'AV 'G KV M V L jPRSAAVKRGR KrLMDVVPLAPRSAAVKRGR KFLMDVVPLA

PRSAALKRGR KrLLDVVPLAQRSAAQNRGR KYFLDMM LF7 -- - 57 - .* 878- 88 . -6

-URVPGLQVW-KRVPGLQVW

-NRVPGLQVW-ANMALLLN

,jPRKLRLQPW67 445-" 5*

KVT;FTG.CVV

:VT'PTGI:VVEVT FTGCVVKVTFrTGCVA4'LAYYGCA

TgGT1_226380 -M3)LTVSX

TgVEG_226380 MY ' TUTEI

TgME49_2263800 M 06TV1Y

HHA 226380 -- ' VTVQSD (NCLIV_046580 MMVPLITYPINCLIV__047520 MQLPX -LYVV

03IATNQVVA AAAKSUSII fESTGIGESAE

R3IATEQVVA AAAKSTS XT STGIGEAE

RGIATEQVVA AAASEE:E TGIGEDAE

GTATEQVVA AAA-RSE-T GPSRERVLATRQVNA ADAS--.---Z jiAGXPVPAG

HGM--EDAGN SRPSEPGAGT GGTLFQNEPS

FMRELVXVREEHRELVKVRE

'EHRELVKVRE

EHRELVXVRE

EIRQELTETRQ.ZEXEEMRE

. . .. .,. 9 .... .)) . . .. . o . . .5.. . 3

TgGT1_226380 ASPVNRPRPE PPOD -AN SVAVANTOIP RYGDASVDD- RGLARVT *RTDPD R EE3L0A

TgVEG_226380 ASPVNRPRPE PP GDDAN SVAVANTSP RYGEASVD- Vf6OLA05T RPTIP0O EEGLOQA

TgE49_226380ASPVN ItPS PPGD AN SVASANTIIP RYGEASVD- 11 SRGLARV 3T ) RTDPD Q ASEVERLYQA

HHA_226380 ASPVNRRTPI PPPVD SUAN S1AE 1T!VP SREZATVDD- UERRGLARVT IV RTEPDKAK ASEV'RE1.RYQA

NCLIV 046580 SSAWDLREK QPSAAGK AD SALVASTRVP -- SDYEDA TPPROLARRT LWGTNA-ALQ APEAGRLDEG

NCLIV_047520 VSEPNIAK-A 6P--- - N SAGTTTS- - ---DDLSEG- VKKDDTAAPR YPLTEEEEAD AVERGRIAE A

XQRQNAEAQWKQRQNAEAQWQERQOAEAQW

KQRQKAEEQKMQARASQEELSQRMVASTIE

00KC(QQLQPEXtFWQQLQPE

KFWQQLQPE E

KHFWQQLt'D 4RRFWNRI.HPHEDPLNRrLAC

SDLATAAAEX GNKDE--4 LATAAA G1D-

SELDAAAAEO GNID -

0DL AAAAEI GRIDV--

ADFVTAIGIL HPQVRHLPRRDL ,NASNIXI AIRE ST KL-GK

EALRGQLLPEALPGQLLP

EEALRGQLLP

EEALUSQLLP

EEAVRSSAL.P

R D R1,RTT LTrP

I I L1GE41

DVVRLEG;EVR

DvvRLEGEVK

EVVRLESEVR

G3vVSLEQE 1ENLVRLESELA

(PRED) TgGT_22630 P0PRL8ESQR(PRED) TgVEG_226380 PR PRLRKTQR

(PRED) TgME49_226380PRPRLESQR

(PRED) HA 226380 PRPRFRKSQR

(PRED) DCLIV_046580 -- LAIRKPRV

(PRED) NCLIV 047520 LVSMTXV3 KS

RSRSLSLRQR LE1.XVGA - WVGAAAr KTWYARQ.

RSRPLSRRQR LLYVGAV V0AAA KTWYAKRA--

P3S0LSRRQR LLYKVGAVC -VGAAIA P KTWYSKRQRANTLSRRQR LLYKVGTVV1L A-VVGAAVAL KAWYSRRQNT3N-SRRRQ LRIGMAV. A-IVGAAMIV KAAYSRR -

P G-'A RSWL KRWVYAAGVAA kTLLGAYAF RKARRRRNN

BARDRDLPSL

WARD R DLP 5L)EARDRDLPSLEARDRDLPSL

EARARDLPTL

EAZRRSSPTW

SKLYEQLKE

SMLYQQLKES6LYQQLEQ

STLYKQLORPSLYQQ14EEMLLADQLVE

A NRRRVVSTVD DQLERLNRQI.

E NRRRVVSTVP DQLERLNRQL

A NRRRVVSTVD DQLERLNRQ

A DSQRVVTTLN DQLGRLNRQ

A RGQfTSTLD EQITALNRQVk GRT A K RLQ SYVGEVVBDIL

(PRED) TgOTl_226380 YLREAFCY T.CGRADVAQA LVDGLLTERD KVNIDINRAR QEES3AGTA AEEHILYT3L PLRG-PAT.VR MSEDVLRQVQ(PRED) TgVEG_226380 YELRERArCY TRGR3ADVAQA LVDGLL7ERH VIIRAR QEE5EA;(GTA AEEHILYTL PLRG- PAVR MSEDVLRQVQPRED) T E49_22630YELRERAFCY TRGRADVAQA LVDGLLTERH KVNILINRAR QEE3EA3G0TA AEERILYTQ PLRG-PARIO MSEDVLRQVQPRED HHA_2263D0 SALLARAFCY TGRADVAQA LVGGLLTERH DVNVLINRAR NSEEEAKGTA AEEHIL PPRA PAYVR OAQEAIDQV

PRED) NCLIV 0465 GSASA ALG LV3 V SSTDT PSRSEPGILAaADRQ ANPRED) MCLIV_047520 sG P IPN ETP GGQE-PQ LR

B. B ~ 0. .. .. . .. ... .I0(PRED) TgGTI1_237015 M-TVP PCPERRRS

(PRED) TgME49_237015 M-TVP C CPERRRS

(PRED) TgVEG_237015 M-TVP FCPERSRRS(PRED) HHA 237015 0-AVPDDVMG VFCPERSRRS(PRED) NC_BN1204_05091MVEPULG IPCPERPRRN

KRHDRPTVHLNRHDRP I VELNIZH ,RPIVHLNRHDRLTMRLDRTS:PTLRV

.... 61...... 0',

(PRED) TgGT1_237015 C---EAANDM DmSDGGH SAOLCEDIAQ(PRED) TgE49_237015 C---AA.D D SDGGHE SAOLVEDIAQ(PRED) TgVEG_237015 C---AAED6 Dp DGGHE SAOLVEDIAQ

(PRED) HA_237015 C - - -CDADT SEGHD SAG. SDI AP

(PRED) NC_BN1204_05091CAVP SAASV-GDV EDTPG

CALSLVCLIC

FALSLVCLICFALSLVCLICrALSFVCLTC

LSCF'LVLTGH

LSCFLVLTG:!i

L.SCFLVLTG.;i

LTYPLVLTGil

LYCF9VSTGH

GHKTRSGLDE STSTTSRQRR

GHRTRSGLDE STOTTSRQRR

OHKTRSGLOE STSTTSRQRO

GHKTRSDb SASTTSRQRRGHKEQSFPSD N33730E? R

.........1 ( 0FFRSRENLEA

FRSRENLEAPFRSRENLEAYER-RETLTDTYRPQOVYLTI

ARSRLREQ- -

ARSRLREQ-- -

ARSRLREQ--- -

ARARFREQ-- -

ASAVLAVVGD D

I . . . . . . . . 17W. . . . . . . . 180 . . . . . . . . . 190.TVAETAATVQ ENTRRPFRWR DAPEPTSRAE

YVAETAATVQ ENTRRPFRWR DAPEPTSRAE

YVAETAATVQ EETRRPFRWR DAPEPTSRAE

YVAETVSAVD EWTRRRLQPR DVPEPTPRAE

DLSTASRGVW QOWIRGRRTRA DAGG--GEAA

--- AQDHV

-- AQDHG

-- AQVHG- - -VQERTRR G*IOV

220.. .. .. 230.. .. .... 240.

* SVRTGLDA-R GVPMVPRLPP

* PGRTGLDA-R GVPMVPRLPP

* PGRTGLDA-R GVPMVPRLPP

A AVRWGLEAPE GGIPREPP

Q PELTGRT--E GDPFGPV--P

I 0. . . . . . . . 12, . . .3 I ... .I ...........263. ........27 1.(PRED) TgGT1_237015 * OVOV(WAQSSERP PRSAAVRG KLMDVVPDA AVLAICVALR N--NRVPGL WDRPPEYTD GGAPGSG

(PRED) TgRE49 237015 RVY RFS VSQSSERP PRSAAVCRGR KPLMDVVPLA AVLAICVAPR W- -j PGL WDRPPEYTD GGAPGSG

(PRED) TgVEG 237015 YRRFS VS;QSSERP PRSAAVKRGR EFLMDVVPLA AVLAICVAFR -- NRVPGL WDRPPEYTD GGAPGSG

(PRED) HHA 270 15 9 itCP ;t SwQSSERR PRSAALKRGR KFDLDVVPLA AVLAIFVAFR R--NAN DQPPPYTH RGPPGSG

(PRED) NC BN1204 05091 KHGR RG TLIPSELQ PWHLPPRYEA -- PPDQG

C.(PRED)(PRED)(PRED)(PRED)(PRED)

(PRED)

(PRED)(PRED)

(PRED)(PRED)

TgGT_236870 DAA----ASS FLWPPVRRQQTgVEG_236870 MAA----ASS FLWPPVRRQQTgME49_236870AA----ASS FLWPPVRRQQHHA_236870 OAA----ASY FPWPPVRRQLNCLIV_050730 MAALPSSPRS LAWP -----RS

TgGTI_236870 PFQ LG PPAS

TqVEG 236870 PFQ LGFP-A'

TgME49_236870PFQ _ LGFPPAS

HA_236870 PFQQTDTRAS PSNLSFGPASDCLIV_050780 ITQRRDTGAS SSELGFLPVP

(P.E. 120.

(PRED) TgGT_236870 DGKVLEPVHA DSKEEELDDD(PRED) TgVEG 236870 DGRVLEPVHA DSKEEE2LDDD U

(PRED) TM4 9_236870 DGRVLEPVHA DSKEEELDDD

(PRED) HHA_236870 DOVQEET D3M0EEEPDDD

(FRED) NCLIV_050780 TRRSQ3TRNT DSQIINBYDDE Q

LPQRLRPFLHLPQRL.RPFLN

LPQRLRPFLHLPQTLRPPLH

LPLKLRPFDPR

SAMLLGDSPH DSAMLLGDSPH DSAMLLGDSPH D

RAMLLGDPPY V

PPATL.GDSPD D

. .. . . jEDESI ,PVD 0EDES VPVDEDES .VFVD

EDESVSVPD

QEEPVS IPVD x

KVTFFTGCVV SLIFQVGVAA

DVTFTGCVV SLIFQVGVAAKVTFFTGCVV SLirQVGVAAKVTFFTGCVA SLIFQVGVAAXILAFFTG;CVA SVlSQLGVkA

DTPGH3PL 0

TPGCE LDTPGRE L G

VTPGHEENKL GDTPGHGGNKR

. 1411.KRLAKHYRLPKRLAXI1YRLP

KRLARMYRL P

KRQAKHYRF P I

IRVAARTRFP

GAHRRTSDSL

GAHRRTSDSL

GAHRRTSDSL

G&RRRTS109L

GTDGNASY SG. .I ...1511LD* DUTRRYPLD: jTRRYPLD ;TRRYP

LDAVDTRATQ

LoVDTRRS P

. . . . . . . . . 160. . . . . . . . . 170, .. . .. . . . . . . . 190. . . . . . . . . 20CITKKAGN M DMIPGG LTVGVLArLL WQRMAPPQL PD-FVP-ALR

HTKKAGN DMIPGG LTVAVLALLL WQRMAPPQL PD-FVP-ALR

HTKKAGN DMIPGG LAVAVLALIL WQRHMTPPQL PD-FVP-ALR

DAKAGNRV DMIPGA LTVTILALIL WQQRTAPLRM PD-VDE-" L

STKKGGMANK DLLLDTFPGLD VAVTILGL-L RRLSEAEEG PAPEEPL

PGQQLPPP-Y EPWTRAAANP N---------PGQQLPPP-Y EPWTRVAANP N -- --

PGQQLAPP-Y EPWTSVATNP N--PG--L-PP-Y EPWRRVASSP N---------YG --- PPPVY EPWRRVARRP RDDINVTQRP

Figure 5. TgGTI_226380, TgGTI_237015 and TgGTI_236870 have transmembrane domains andseveral alpha helices. PSIPRED was used for secondary structure prediction (Jones 1999). Helices, red.Strands, blue. TmiHMM2.0 was used for transmembrane domain (TM) prediction (Krogh et al. 2001). TMare marked with black lines above region, mutations marked with red asterisks above mutated base.

I153

I

A.(PRED)(PRED)(PRED)

(PRED)(PRED)

(PRED)

(PRED)(PEED)

(PEED)

(PEED)(PRED)(PRED)

PHIVGMDPDT

PHIVGMDPDt

PHMVGMDPDE

PHIVGMDPDS

PNFVGVDPDI

PSTVGM--S

AEPKQT ----

AEPKQT---

AEPKQT -----

VDPKGT ----

QVKTGT ----TPPVSDSDPH

....... . 2040GTEEEDADTA

GTEEEDA DTA

GTEEEDADTA

GTEEEEAEIA

AVTSAQLRAA

TTEETXRGSATYEETHRD)SATTEETHRIDS&T YAEAMRDSGSYEETVNRSH

ALE !2LE

ALEDFDLLLE

ALEDrDVLLE

ALEzDFDVLLE

ALEVKVDALLAG;LErMTVC;MLE

LAVVaGLVCLAVVULSLVCLAVVGLSLVC

LAVVGLNLVC

LAAVAIGrVC

VG;VVIARIEV

30 .. . . . . 41) . . . . . . . . ;0

S

P

We C-terminally tagged each gene product with a hemagglutinin (HA)-tag and confirmed

expression of the protein using immunofluorescent microscopy and Western blot (Figure 6A).

We performed microscopy studies to determine the subcellular localization of the each protein.

The protein products of TgGT1_226380, TgGT1_237015 and TgGT1_236870 each colocalized

with the dense granule protein, GRA7 and therefore were named GRA 18, GRA27 and GRA28,

respectively (Figure 6B).

A- GRA18 GRA28 GRA27 B.

Ex In Ex In Ex

37kD- HA uu ur25kD-20kD-

.l- SAG-iU.-.a o .25kD-

A

an~ y sea N oc verge

Figure 6. TgGTI_226380, TgGTI_237015 and Tg-GTi_236870 are dense granule proteins. (A and B)Strains individually knocked out in each gene were generated using CRISPR/Cas9 and complementedwith HA-tagged wild-type version of gene. (A) HFFs were infected with HA-expressing parasites for 36hours. Extracellular parasites were removed and washed with PBS prior to lysing ("Ex"). Remaininginfected cells were lysed ("In"). SAG-I is used as parasite loading control. Predicted size: GRA18,42.6kD. GRA27, 29.3kD. GRA28, 23.8kD. (B) HFFs were infected with strains expressing HA-taggedGRA17, GRA27 or GRA28 for 24 hours and subjected to IF with antibodies indicated. The imagesrepresent single deconvoluted focal slice. (scale bar = 10 pm).

Primed BMDMs were more sensitive to parasite-induced pyroptosis with strains deficient

in GRA18 GRA27 or GRA28 compared to unprimed macrophages. We did not see this

difference in primed macrophages infected with wild-type or complemented strains (Figure 7A).

To confirm the mutation of these genes were responsible for the failure to activate the

inflammasome in our chemically mutagenized parasites, we expressed the wild-type allele of the

154

gene in each mutant. Addition of wild-type GRA18, GRA27 and GRA28 in their respective

mutant was sufficient to restore pyroptosis induction (Figure 7B). Macrophages primed with

LPS were more sensitive to infection with mutants (Figure 1E, 7C).

A. 120 M - LPSA 12 0- LJ+ LPS

10

>, 80

60

40-

20.

0.

C N

B.*** **** ****

100

:80

Q60-

40-

20-

02

x x . xc~ x

120 - LPS

+ LPS10

80-

60

4-

2

01HC

Figure 7. Addition of wild-type version of gene is sufficient to restore ability to induce pyroptosis.

(A) Lewis BMDMs were primed with LPS (l00ng/ml, 2-4 hours) or left untreated and infected with

indicated strains (MOI =1, 24 hours). Viability measured by MTS assay. Graph is average of 2

experiments. Error bar, + SD. (B) Lewis BMDMs infected with indicated strains (MOI =1, 24 hours).

Viability measured by MTS assay. Data is average of 3 experiments. Error bar, + SD. ***p<0.0005,

****p<0.0001. (C) Lewis BMDMs were primed with LPS (100ng/ml, 2-4 hours) or left unprimed and

infected with indicated strains. Data is I experiment. Error bar, + SD. Data is from I experiment.

155

As expected, 80% of macrophages infected with RHAGRA18, RHAGRA27 or

RHAGRA28 contained multiple parasites, whereas upwards of 60% of wild-type and the

complemented strain-infected BMDMs contained only single parasites (Figure 8A). Similarly,

only ~30% of macrophages infected with mutant strains expressing wild-type GRA 18, GRA27

or GRA28 allowed replication of Toxoplasma, in contrast to ~80% of mutant-infected BMDMs

(Figure 8B).

To determine if complementation of the mutants was sufficient to restore IL-10 cleavage

and activation, we infected LPS-primed BMDMs and measured IL-1 secretion. Infection with

RHAGRA18 or RHAGRA28 resulted in a significant decrease in secreted IL-i$ compared to

wild-type and their complemented counterparts (Figure 8C). Expression of wild-type GRA18,

GRA27 or GRA28 in mutant strains was sufficient to induce a significant increase in IL-ip

released from primed BMDMs (Figure 8D). When we probed for active IL-ip, we observed an

increase in the active 17kD fragment secreted from macrophages infected with the

complemented strains compared to their mutant counterparts (Figure 8E). Additionally, primed

BMDMs infected with ME49AGRA27 and ME49AGRA28 secreted less active IL-1p than wild-

type infected cells (Figure 8F).

156

A.100-

-) 80-

60-a)

40-

20-

C. 4000 -

3000-

0)2000-

1000-

I-T

Hl7'

>11

parasites/vacuole

*** *

U 9== -

0 0

C, X

+LPSE. N

X X

37kD-

17kD- ma

MW-T B.[i]AGRA18 100.ELAGRA18 + GRA1 8E-]AGRA27 =T 80 -

[-]AGRA28 aMAGRA28 + GRA28 a)

40.

20 IP-wr[ZMutant#1[llMutant #1 + GRA28P~ LlMutant#2

MMutant #2 + GRA27Mutant #3

MMutant #3 + GRA18

>1parasites/vacuole

D.

: 3000-

2000-

1000-

Oii*0N~~f

N~

F.Toxo - - WT AGRA28 AGRA27

LPS- + - + - + - +

37kD-

17kD-

Figure 8. GRA18, GRA27 and GRA28 are required for inflammasome activation. (A and B) Number

of parasites per vacuole of infected Lewis BMDMs (MOI = 0.5, 24 hours) as measured by microscopy.

Between 50-100 vacuoles were scored per experiment. (A) is average of 4 experiments. (B) is average of

2 experiments. Error bars, + SD. P-values are <0.01 comparing mutant strains to wild-type or

complemented strains (Two-way ANOVA). (C and D) IL-1f as measured by ELISA from LPS-primed

(100ng/ml, 2-4 hours) Lewis BMDMs infected with indicated strains (MOI = 1, 24 hours). Data is

average of 3 experiments. Error bars, + SD. *p<0.05, **p<0.001, ***p<0.0005, (E) Western blot of IL-

IP on concentrated supernatants (25X) BMDMs primed with LPS (100ng/ml, 3 hours) infected with

indicated strains (MOI =1, 24 hours). Image is representative of 2 experiments. (F) Western blot probing

for IL-1f on concentrated supernatants (20X) of Lewis BMDMs that were primed (100ng/ml, 3 hours) or

left unprimed and infected with indicated strains (MOI =1, 24 hours). Image is representative of I

experiment.

157

T TT

4q=PW,_6_

Rn I

U

Strains deficient in GRA18 or GRA27 do not establish chronic infection in Lewis rats

In chapter three, we hypothesized that Toxoplasma utilizes the macrophage as a vehicle for

dissemination away from the initial site of infection to distal sites, including the brain.

Activation of the NLRP1 inflammasome in Lewis rats results in pyroptosis. Host cell death

results in the destruction of the niche Toxoplasma requires for proliferation and trafficking

throughout the body. In chapter four, we identified three parasite genes, GRA18, GRA27 and

GRA28, required for activation of the NLRP1 inflammasome and pyroptosis in macrophages in

vitro. We hypothesized strains deficient in these genes will fail to induce pyroptosis in

macrophages in vivo, allowing the parasite to replicate and move to the brain. Parasites lacking

GRA18, GRA27 or GRA28 would then be able to convert into the dormant bradyzoite stage and

establish a lifelong chronic infection.

To test this, we infected Lewis rats and Brown Norway rats with wild-type ME49-RFP

(ME49), ME49AGRA 18 and ME49AGRA27, intraperitoneally. RH parasites do not readily form

orally infectious cysts in mice or rats, while the ME49 strain does (data not shown). After four

weeks, we harvested brains and determined the presence or absence of cysts via rederivation in

vitro. Preliminary results suggest that individual deletion of GRA18 or GRA27 is not sufficient

to establish a chronic infection in Lewis rats. Wild-type ME49 parasites were able to form cysts

in the control Brown Norway rats, in which Toxoplasma readily establishes a chronic infection

(Sergent et al. 2005) but not in Lewis brains. Neither ME49AGRA 18 nor ME49AGRA27 were

rederived from Lewis brains. Zero Brown Norway rats infected with ME49AGRA18 contained

cysts within brain tissue. One out of two ME49AGRA27-infected Brown Norway rats contained

cysts (Table 2).

158

Number of rats with detectable cysts

Strain Brown LewisNorway

Wild-type ME49-RFP 2/2 0/2

ME49-RFPAGRA 18 0/2 0/2

ME49-RFPAGRA27 1/2 0/2

Table 2. Lewis rats infected with ME49AGRA18 or ME49AGRA27 are not chronically infected.Lewis and Brown Norway rats were infected with 3 x 106, i.p. for 30 days. Brains were harvested andbrain suspension was placed onto HFFs for parasite rederivation. Table is one experiment.

The failure of the ME49AGRA18 and ME49AGRA27 to form cysts as well as wild-type

in the susceptible Brown Norway rats suggests that these genes are required for establishing a

chronic infection. To successfully infect a host chronically, Toxoplasma must replicate, traffic to

distal sites, sense an immune response and convert from the quickly dividing tachyzoite into the

semi-dormant bradyzoite. To determine if parasites lacking GRA18, GRA27 or GRA28 are able

to replicate properly, we measured plaque area formed in human foreskin fibroblasts (HFF)

monolayers. Measuring plaque area accounts for the overall growth of parasites in vitro,

including parasite replication, egress and reinfection. We found no significant differences in

plaque area between HFFs infected with wild-type parasites and RHAGRA18, RHAGRA27 or

RHAGRA28 (Figure 9). This data, in addition to our findings that RHAGRA 18, RHAGRA27

and RHAGRA28 are able to replicate within the Lewis macrophage suggest that the absence of

these genes do not play a role in parasite growth.

159

500

400

E 300-

2 200

100

0

Figure 9. RHAGRAI8, AGRA27 and AGRA28 do not show a growth defect in vitro. Confluent HlFFswere infected with the indicated parasites for 5 days. The area of at least 30 plaques per experiment wasmeasured. Data is average of 2 experiments. Error bars, +v SD.

GRA27 and GRA28 activate type I interferon response pathways

Toxoplasma must activate the immune system in order to convert from tachyzoite to

bradyzoite. Several Toxoplasma proteins have been established as regulators of host gene

expression, including immunological pathways. We were interested in determining if GRA 18,

GRA27 or GRA28 act as modulators of host gene expression and performed RNAseq on Brown

Norway BM4DMs infected with our knockout and complemented strains. 1 72 transcripts were

differentially regulated by GRAI8, GRA27 and/or GRA28 (Figure 10A, Table 3). Gene

ontology (GO) analysis found immune system process (p-value =6.31 x l0-8) pathways as

significantly enriched among these transcripts (Figure lOB).

160

Among the genes differentially regulated were several type I IFN-regulated genes,

including Glp2, which encodes for a ubiquitin-like modifier protein, Ifitm3, encoding for an

interferon-induced transmembrane protein and IrJ7, a transcription factor typically activated by

TLR3, TLR4, TLR7 and TLR8 (Schoenemeyet et al. 2005; Doyle et al. 2002). GRA27 and

GRA28-complemented strains induced greater expression of Glp2, Irf7 and Ifitm3 in Brown

Norway macrophages than their mutant counterparts (Figure 10C). Interestingly, macrophages

infected with GRA18-deficient parasites expressed these genes to a lesser degree than the

complemented strains, suggesting GRA18 may act as a negative regulator of the interferon-

response.

We observed a two-fold reduction in expression of Ilib, which encodes for pro-IL-i IP, in

macrophages infected with RHAGRA28 compared to those infected with its complement (Table

3). Unlike pro-IL-18, pro-IL-1 is not constitutively expressed in myeloid cells. Inflammasome

activation and release of active IL-1p requires two distinct signals: priming and recognition of an

agonist by the sensor. Toxoplasma supplies both signals. The mechanism through which GRA28

induces pro-IL-ip expression should be explored further. Interestingly, GRA28 also leads to

increased I1r2 expression. This gene codes for the IL-I receptor, type II (IL-i R2), which acts as

a decoy IL-i receptor (Colotta et al. 1993). IL-IR2 binds IL-1, but does not signal. Toxoplasma

activation of IL- IR2 may be a mechanism the parasite uses to prevent excessive IL- 1$ signaling,

which can lead to IFN-y production and parasite clearance.

161

respon*ytokine

response to stimulus

response to biotic stimulusregulation of cell proliferation

multi-organism proces

negative regulation of biological process

response t organismnegative regulation of viral genome replication

A. -o

0 0'00 E

0 0 C.

E E0 tot o

-CO C\D Cn CDD CZ CJ C'4 (,J

response to external stimulus

C- 150

e 10&CLU-

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('C,

USO

500-

400-LL

ii 300

-'200-C\.JI-100

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100

80

CY)

E40-

20

0- -

Figure 10. GRA18, GRA27 and GRA28 are involved in IFN-regulated signaling. Brown Norway

BMDMs were infected with indicated strains for 18 hours (MOI 1). Strains used were RHAGRAI8,

RHAGRA18 + GRA18. RHAGRA27, RHAGRA27 + GRA27, Mutant #2 and Mutant #2 + GRA27. Total

RNA was extracted and subjected to RNAseq. Differentially regulated genes were identified after

normalization and filtering genes that had at least a two-fold difference in expression between strains.

Data is one experiment. (A) Heat map of differentially expressed rat genes. (B) Visualization of gene

ontology analysis of genes in Table 3. Map created with REVIGO (Supek et al. 2011). Intensity of red

indicates p-value, size of bubble indicates the frequency of GO term and line thickness between nodes

indicates similarity. (C) Gene expression of representative interferon-regulated genes.

162

immune system process

imrniune response

B.

cell surface receptor signaling pathway

defense response

GeneLOC252890OC15CCIIOCXCI1 1GlIo2SItn 1L0C6891 66Ititm3Lv6eIrf7RT1 -T24-3LOC100363494LOC100360657sa 12(b)

SbsnLOC100361539KIra2LOC100361633Rbo2RGD1564744L0C680693LOC100360357Tpm2

w -~

Gene Description Log2 Fold Change

GRA18 GRA27 GRA28

LOC100361596 Hypothetical protein 3.17 -9.37 -1.66

LOC100361060 Ribosomal protein L36-like -1.4 -8.21 3.16

LOC64038 Sertolin -2.2 -5.35 -0.74

Psma3l Proteasome subunit alpha type 3-like 7.83 -3.15 -2.59

rCG_65891 Hypothetical protein 0.63 -2.75 -0.7

Ccl5 Chemokine (C-C motif) ligand 5 0.72 -2.27 -2.23

Cxcll 1 Chemokine (C-X-C motif) ligand 11 0.05 -2.21 -1.9

Cxadr Coxsackie virus and adenovirus receptor -0.86 -1.82 -0.71

LOC100362623 Hypothetical proteins -0.5 -1.81 -1.17

Mt2A Metallothionein 2 -0.46 -1.78 -1.22

LOC100360841 Ribosomal protein L37-like -0.62 -1.77 -0.26

LOC 100360499 Guanine nucleotide binding protein -7.9 -1.77 6.89

Mtla Metallothionein 1 -0.82 -1.71 -0.55

RGD1560592 Hypothetical protein -0.42 -1.62 0.33

LOC689166 Hypothetical protein 0.79 -1.57 -1.23

LOC 100360773 Hypothetical protein 0.9 -1.55 -0.49

CclI Chemokine (C-C motif) ligand 1 0.11 -1.52 -1.59

Rsad2 Radical S-adenosyl methionine domain containing 2 1.83 -1.52 -0.87

Vwal Von Willebrand factor A domain containing 1 -0.22 -1.49 -0.83

Gjb2 Gap junction protein, beta 2 -0.46 -1.45 -0.81

Glp2 ISG15 ubiquitin-like modifier 0.27 -1.43 -1.91

Mx2 Myxovirus (influenza virus) resistance 2 1.51 -1.4 -1.55

Sdcbp2 Syndecan binding protein (syntenin 2) -0.9 -1.4 -1.02

Fam89a Mammary tumor virus receptor 2 isoform-like -0.55 -1.4 -0.91

Tnfsfl 8 Tumor necrosis factor (ligand) superfamily, 18 -0.28 -1.39 -1.54

rCG_32844 Ubiquitin specific protease 43 -0.79 -1.27 -0.21

Kcnk5 Potassium channel, two pore domain subfamily K -0.58 -1.26 -0.08

Egr4 Early growth response 4; transcription regulation -0.15 -1.25 -1.13

Rasl 1 Ia RAS-like family 11 member A -0.3 -1.25 -0.81

Procr Protein C receptor, regulation of coagulation -0.6 -1.25 -0.57

Arc Activity-regulated cytoskeleton-associated protein -0.44 -1.24 -0.45

Ly6e Lymphocyte antigen 6 complex, locus E 0.82 -1.21 -1.58

Egfl7 EGF-like domain, multiple 7 0.11 -1.19 -0.53

Uap112 UDP-N-acetylglucosamine pyrophorylase 1-like 2 -0.38 -1.19 -0.49

Zdhhc2 Zinc finger, DHHC-type containing 2 -0.62 -1.18 0.16

163

AssI Argininosuccinate synthase 1 -0.15 -1.14 -1.21

Faml32b Complement Clq TNF-related protein -0.53 -1.14 -0.81

LOC689442 Hypothetical protein -0.82 -1.13 -1.08

Hap] Huntingtin-associated protein 1 -0.5 -1.13 -1.05

Uppi Uridine phosphorylase 1 -0.48 -1.13 -0.98

Asgr2 Asialoglycoprotein receptor 2 -0.3 -1.13 -0.94

Rasdl Ras, dexamethasone-induced 1 -0.31 -1.13 -0.65

Clcfl Cardiotrophin-like cytokine factor 1 -0.12 -1.13 -0.58

Wispl WNTl inducible signaling pathway protein 1 -0.3 -1.12 -1.07

Gfpt2 Glutamine-fructose-6-phopshate transaminase 2 -0.52 -1.11 -0.78

Tnfrsf9 Tumor necrosis factor receptor superfamily 9 -0.46 -1.11 -0.09

Efnb2 Ephrin B2 0.34 -1.1 -1.06

Slfn3 Schlafen 3 -0.01 -1.09 -1.44

Mxl Myxovirus (influenza virus) resistance 1 1.32 -1.06 -1.53

116 Interleukin 6 0.05 -1.06 -1.42

Cxcl2 Chemokine (C-X-C motif) ligand 2 0.06 -1.06 -1.24

Cxcl Chemokine (C-X-C motif) ligand 1 0.03 -1.06 -1.01

Phlda2 Pleckstrin-homology-like domain, family A, 2 0.07 -1.03 -1.2

Mbnl3 Muscleblind-like splicing regulator 3 -0.42 -1.02 -0.32

Btg3 BTG family member; RNA degradation pathway -0.37 -1.01 -0.49

LOC100363494 Ubiquitin B-like 2.18 -1 -3.44

CxcllO Chemokine (C-X-C motif) ligand 10 0.44 -l -1.27

Tjp2 Tight junction protein 2 -0.42 -1 -0.8

Bspry B-box and SPRY domain containing -0.22 -0.98 -1.38

Cdc42ep2 CDC42 effector protein (Rho GTPase binding) 2 0.1 -0.98 -1.24

Gprc5a G protein-coupled receptor, class C, group 5 -0.38 -0.96 -1.15

Fgf2 Fibroblast growth factor 2 0.21 -0.95 -1.38

RGD1564664 Hypothetical protein -0.29 -0.94 -1.79

Gimap7 GTPase IMAP family member 7 0.39 -0.92 -1.77

Pcskl Proprotein convertase subtilisin/kexin type 1 0.01 -0.89 -1.21

Oasla 2'-5' oligoadenylate synthetase IA 1.06 -0.87 -1.15

RT1-T24-3 RT1 class I, locus T24, gene 3 0.91 -0.82 -1.44

Irf7 Interferon regulatory factor 7, transcription factore 1.05 -0.79 -1.47

Gbp5 Guanylate binding protein5 -0.02 -0.79 -1.16

Hspa2 Heat shock protein 2 -0.23 -0.73 -1.08

Ifit3 IFN-induced protein with tetratricopeptide repeats 3 1.63 -0.67 -0.97

Penk Proenkephalin 0.02 -0.66 -1.62

164

LOCIO0360640 Pxtl; positive regulation of apoptotic process -2.91 -1.17 -0.09

Scin Scinderin; Fc receptor mediated signalling -0.05 -0.64 -1.1

Sbsn Suprabasin -0.42 -0.62 -1.14

Slfnl Schlafen 1 0.29 -0.61 -1.28

Lgals3bp Lectin, galactoside-binding, soluble, 3 binding protein 0.69 -0.6 -1.01

Nupri Nuclear protein, transcriptional regulator, 1 -0.04 -0.59 -1.34

SiglecI Sialic acid binding Ig-like lectin 1, sialoadhesin 0.79 -0.57 -1.08

isgl2(b) Ifi2712b; IFN alpha-inducible protein 27 like 2B 0.32 -0.56 -1.37

Klrkl Killer cell lectin-like receptor family K, member 1 1.06 -0.55 -1.3

Tnntl Troponin T type I 1.02 -0.53 -0.98

RGD1306074 Hypothetical protein 1.12 -0.52 -0.51

Illb Interleukin 1 beta -0.32 -0.44 -1.53

LOC100360657 Hypothetical protein 0.48 -0.42 -1.27

Uspi8 Ubiquitin specific peptidase 19 1.04 -0.42 -0.51

Ifi27 Interferon alpha-inducible protein 27 0.36 -0.41 -1.1

Ddit4 DNA-damage inducible transcript 4 0.16 -0.4 -1.43

Niacrl Hydroxycarboxylic acid receptor 2 -0.18 -0.4 -1.04

FoslI Fos-like antigen 1 -0.52 -0.36 -1.02

Trim471 Tripartite motif protein 80 -0.15 -0.34 -1.1

Klra2 Killer cell lectin-like receptor subfamily A, 2 0.56 -0.33 -1.44

Rem2 RAS (RAD and GEM) like GTP binding 2 -0.54 -0.32 -1.37

Plscrl Phospholipid scramblase 1; regulator of Fc receptor -0.11 -0.3 -1

Neurl3 Neuralized E3 ubiquitin protein ligase 3 -0.13 -0.28 -1.07

Ifit2 IFN-induced protein with tetratricopeptide repeats 2 1.02 -0.28 -0.46

Mgmt O-6-methylguanine-DNA methyltransferase 0.2 -0.27 -1.18

Clec4e C-type lectin domain family 4, member E 0.52 -0.26 -1.88

Prodh2 Proline dehydrogenase (oxidase) 2 0.37 -0.2 -1.42

RGD 1560451 NADH dehydrogenase (ubiquinone) 1 alpha 4.02 -0.2 4.77subcomplex, 7-like

Cd209c Mannose binding, participates in measles infection -0.6 -0.19 1.29

rCG_61839 Hypothetical protein 1.53 -0.18 -0.08

Oaslb 2'-5' oligoadenylate synthetase 1B 1.36 -0.18 -0.06

ArgI Arginase 1 -0.91 -0.17 -1.02

Illr2 Interleukin 1 receptor, type II 0.23 -0.13 -1.38

ttc2 1 a Tetraricopeptide repeat domain 21 A -1.94 -0.12 -1.94

rCG_58138 Hypothetical protein -1.03 -0.11 1.44

Fcgr3a Fc fragment of low IgG, low affinity II1a, receptor 0.72 -0.08 -1.21

165

Ifitm3 Interferon induced transmembrane protein 3 0.7 -0.64 -1.2

LOC686066 60S ribosomal protein L38-like 2.16 0 5.98

Id4 Inhibitor of DNA binding 4 -0.26 0.01 -1.14

Timp3 TIMP metallopeptidase inhibitor 3 -0.28 0.03 -1.16

Apob48r Apolipoprotein B receptor 0.16 0.03 -1.01

Rtp4 Receptor (chemosensory) transporter protein 4 1.14 0.08 -0.7

LOC691532 40S ribosomal protein S25-like -0.27 0.09 -2.46

RGD1559682 Cyclophilin A-like -0.14 0.1 -1.42

RGD1562462 Similar to interferon activated gene 204 1.01 0.1 -0.41

Trem3 Triggering receptor expressed on myeloid cells 3 -0.23 0.11 -1.24

RGD1562542 Ribosomal protein Sl2-like -0.52 0.13 -1.53

Pcdh8 Protocadherin 8 -0.59 0.13 -1.06

LOC679594 Polyubiquitin-like 5.11 0.13 0.58

nod3l Nod3-like; leucine rich repeat containing 73 -1 0.13 1.06

Ypel4 Yippee-like 4 -1.04 0.14 -0.37

LOC24906 Hypothetical protein 0.67 0.16 -1.89

RGD1564744 60S acidic ribosomal protein P1-like 0.44 0.2 -2.95

Oscar Osteoclast associated, Ig-like receptor 0.38 0.28 -1.52

Tpm2 Tropomyosin 2, beta -0.13 0.28 -1.01

LOC688430 Cofilin-1 0.56 0.29 -1.16

LOC680693 Sperm flagellar protein 1-like -0.1 0.31 -1.03

Lipg Lipase, endothelial 0.48 0.38 1.01

Col3al Collagen type III 0.24 0.4 1.47

Rasgrpl RAS guanyl releasing protein 1 (calcium regulated) -0.04 0.42 1.33

LOC100361633 CD300 molecule-like family member 0.31 0.44 -1.61

Ubd Ubiqitin D 0.92 0.46 2.23

Tgfbi Transforming growth factor, beta induced 0.18 0.5 -1.12

Serpinhi Serpin peptidase inhibitor; heat shock protein 47 0.03 0.53 1.07

Illf8 Interleukin 36 beta 0.39 0.57 -1.19

Cd209a Mannose binding, participates in measles infection -0.7 0.7 1.06

Fcrll Fc receptor-like 1 -1.33 0.73 -0.2

Rbp2 Retinol binding protein 2 0.26 0.76 -2.06

LOC100360357 Cytochrome C oxidase, subunit VIlc-like -7.02 0.8 -3.65

RGD1559951 60S ribosomal protein 37a-like 0.82 0.8 -1.39

Leap2 Liver-expressed antimicrobial peptide 2 1.2 0.82 1.48

Hr Hair growth associated; histone deacetylase binding 0.4 0.83 1.09

Ccl6 Chemokine (C-C motif) ligand 6 0.78 0.85 -1.36

LOC100362983 Small integral membrane protein 7-like -1.43 0.97 0.95

166

LOC252890 Z39 small nucleolar RNA 2.17 0 -14.95

Tmem150 Transmembrane protein 150A -0.06 1.01 0.17

Lpar5 Lysophosphatidic acid receptor 5 -0.34 1.02 0.74

RTI-Ba RT1 class II, peptide antigen binding 0.27 1.07 0.46

RGD1309808 Apolipoprotein L2-lik3 0.52 1.1 0.09

Clec4d C-type lectin domain family 4, member D 0.65 1.14 -1.19

LOC100361539 Immune activating receptor CD300dl-like 0.59 1.14 -1.15

LOC 100362961 Hypothetical protein 0.44 1.17 0.1

RT1-Da RTI class II, MHC class II protein complex binding 0.23 1.17 0.21

Gcgr Glucagon receptor 0.6 1.18 -0.03

RGD 1562406 Histone deacetylase complex subunit SAP25-like -0.51 1.2 -0.08

Pik3ipl Phosphoinositide-3-kinase interacting proteini 0.68 1.25 0.19

Ncr3 Natural cytotoxicity triggering receptor 3 0.26 1.31 -0.1

Cd209e Mannose binding, participates in measles infection -0.58 1.39 1.61

Rspol R-spondin 1 0.59 1.63 0.05

LOC681754 Cytochrome c oxidase, subunit VIb polypeptide 1-like 1.71 1.79 -0.33

LOC680400 Hypothetical protein 0.43 1.79 0.65

LOC100359935 Ribosomal protein L37a-like 1.17 1.8 6.94

Siglec15 Sialic acid binding Ig-like lectin 15 0.82 1.9 0.29

LOC100363339 Hypothetical protein 2.15 2 6.35

FamI9a3 Chemokine (C-C motif)-like, member A3 -0.7 2.1 -0.91

RGD1561465 Hypothetical protein -0.72 2.41 -1.08

rCG_42077 Hypothetical protein -1.88 2.43 1.91

rCG_51406 Neuritin 1-like -0.13 3.68 4.53

LOC100362919 Hypothetical protein 0.09 3.73 -0.21

Table 3. List of differentially expressed rat genes. Data was filtered for genes with FPKM >5in at least one sample and a minimum of 2-fold difference in expression between any mutant andits complement. Log fold change using formula log 2fold = log2(mutant)-log2(complement). Datais sorted by Log 2 fold change of GRA27, followed by GRA28. Data is one experiment.

167

Tslp Thymic stromal lymphopoietin; cytokine activity 0.31 1.01 -1.19

Discussion

In this work, we have identified three novel dense granule proteins, GRA 18, GRA27 and

GRA28, which are required for Toxoplasma-induced pyroptosis and IL-i I activation and release

in Lewis BMDMs. Previous work demonstrated this phenomenon was mediated by Nlrpi

(Cirelli et al. 2014). Several dense granule proteins have been established as players in host

modulation. Type II GRA15 is sufficient to induce host NF-KB nuclear translocation and

activation (Rosowski et al. 2011). GRA16 and GRA24 are exported out of the parasitophorous

vacuole into the nucleus to modulate host gene expression (Bougdour et al. 2013). GRA6 is a

regulator of the host transcription factor, NFAT4 (Ma et al. 2014). The mechanism through

which GRA18, GRA27 and GRA28 coordinate to activate NLRP1 remains to be determined.

GRA 18, GRA27 and GRA28 do not have significant homology to known proteins from

organisms outside of the family, Sarcocystidae, which includes Neospora and Hammondia.

Neospora caninum is able to induce pyroptosis in Lewis BMDMs (Chapter 3 - Addendum,

Figure 1), suggesting the function of these proteins is conserved. Genetically diverse

Toxoplasma strains are able to induce pyroptosis in Lewis BMDMs, supporting this hypothesis.

Interestingly, strains that fail to activate the NLRP 1 inflammasome in Lewis macrophages do not

show a deficiency in activation of the mouse inflammasomes. It is likely GRA18, GRA27 and

GRA28 have other functions important to the success of the parasite and determining these

functions would of interest.

Cleavage of NLRP1 is required for the activation of the inflammasome by Anthrax LT.

GRA18, GRA27 and GRA28 do not have predicted protease domains. They do share predicted

transmembrane domains and several helices (Figure 5). A potential model of activation is

GRA18, GRA27 and GRA28 localize within the parasitophorus vacuole membrane, with C-

168

termini facing the host cytosol, where they mediate a host immune response. This has been

demonstrated by GRA6, whose C-terminus interacts with host cytosolic protein CAML, which

leads to the activation of NFAT4 (Ma et al. 2014). Our finding that individual deletion of these

genes is sufficient to significantly reduce pyroptosis and IL-pI P cleavage supports a model where

the proteins exist within a complex. These proteins may directly interact with or modify NLRP1

or facilitate the recognition of an unknown Toxoplasma protein. Human fibroblasts engineered to

express Lewis NLRP 1 and caspase- 1 or mouse macrophages that express only Lewis NLRP 1 fail

to undergo pyroptosis when infected with Toxoplasma, suggesting that additional co-factors were

required for inflammasome activation (Cirelli et al. 2014). GRA18, GRA27 and GRA28 may

interact and modify a rat-specific protein that is sensed by NLRP1, similar to NLRC4

recognition of a NAIP5/NAIP6/flagellin complex (Kofoed and Vance 2011; Zhao et al. 2011).

We found LPS-primed Lewis macrophages consistently underwent more pyroptosis when

infected with mutant or individual knockout strains. We previously observed LPS stimulation of

Lewis BMDMs induces a two-fold increase in Nlrpi and Caspi and a four-fold increase in

Caspl1. This increase in expression of key inflammasome components may lower the threshold

of activation. Single knockout strains most likely have low levels of the Toxoplasma factor that

is recognized by NLRPL. Generating double and triple knockout strains may further reduce the

available activating factor and prevent pyroptosis completely in primed macrophages.

Preliminary in vivo experiments found individual deletion of GRA18 and GRA27 is not

sufficient to form cysts in Lewis rats. Curiously, these strains show a defect in establishing a

chronic infection in Brown Norway rats, in which Toxoplasma can readily form cysts. There are

several possibilities for this reduction in virulence of these strains. Strains deficient in genes may

not infect cells and replicate at rates comparable to wild-type strains. In vitro experiments

169

measuring growth in human foreskin fibroblasts showed no significant difference in parasite

replication, although in future experiments parasite growth should be measured in Brown

Norway macrophages to confirm this finding.

All rats are relatively resistant to Toxoplasma. While type I parasites have an LDioo = 1

parasite in the common laboratory mouse and will succumb to infection within one week due to

parasitemia, rats can be infected with over one million of the same strain and fail to show

symptoms of an acute infection. This suggests rats have defense mechanisms, which are more

effective at preventing the parasite expansion seen in mice. The host mounts an immune

response upon recognition of Toxoplasma and uses several identified mechanisms to clear

infection, particularly those who are induced by IFN-y. Anti-IFN-y treatment led to an increase

in cyst numbers and antibody titers in Brown Norway rats (Sergent et al. 2005). Parasite ROP 18

and ROP5, have been found to be critical in Toxoplasma's ability to counter the IFN-y-inducible

IRGs (Niedelman et al. 2012; Behnke et al. 2012). GRA18, GRA27 or GRA28 may play a role

in antagonizing a rat defense pathway and deletion of any of these genes renders the parasite

susceptible to these mechanisms.

The "Trojan Horse" model of parasite dissemination hypothesizes Toxoplasma exploits

the ability of leukocytes to traffic to help its movement throughout the body. The intracellular

environment offers the parasite an advantage as it is protected from the immune system.

Dendritic cells, monocytes and natural killer cells infected with Toxoplasma display a

hypermotility phenotype (Harker et al. 2013; Lambert et al. 2009; Ueno et al. 2015). These cells

move faster and across greater distances. Parasite-infected DCs transferred into mice resulted in

faster dissemination of the parasite to distant sites (Lambert et al. 2006). These data support the

"Trojan horse" hypothesis. The ability to induce hypermobility in leukocytes is conserved among

170

strains (Lambert et al. 2009), but the parasite genes involved are unknown. GRA18, GRA27 or

GRA28 may be involved in successful parasite dissemination.

In summary, we have established GRA18, GRA27 and GRA28 are required for NLRP1

activation by Toxoplasma gondii. The identification of these genes raises further questions about

the mechanisms through which NLRP1 is activated and the function of these proteins.

171

Materials and Methods

Ethics statement

All animal experiments were performed in strict accordance with the recommendations in

the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and

the Animal Welfare Act, approved by the MIT Committee on Animal Care (assurance number

A-3125-01).

Reagents

N-ethyl-N-nitrosourea and ethyl methanesulfonate were purchased from Sigma-Aldrich.

CellTiter 96 AQueous One Solution Cell Proliferation Assay was obtained from Promega.

Dextran sulfate sodium salt was obtained from Santa Cruz Biotechnology. Rabbit anti-IL-ip

(ab9722; 1:1000) was purchased from Abcam. Rat anti-HA (3F10; 1:5000) antibody was

obtained from Roche. Secondary HRP-conjugated antibodies were purchased from Jackson

ImmunoResearch. Alexa Fluor 448 and 594 secondary antibodies were obtained from Invitrogen.

Rat and mouse IL-1 DuoSet ELISA were purchased from R&D Systems. Lipopolysaccharide

(LPS) was purchased from Calbiochem/EMD Biosciences.

Animals

Lewis (LEW/Crl; LEW) rats were purchased from Charles River Laboratories

(Wilmington, MA) at 6-8 weeks old. C57BL/6J mice were purchased from Jackson Laboratories

at 8 weeks old.

172

Cell Culture

Tachyzoites from Type I (RH) expressing luciferase and GFP from pDHFR-Luc-GFP

were used for mutagenesis. RH parasites lacking GFP, luciferase and HXGPRT were used for

CRISPR studies. Type II (ME49) engineered to express RFP were a gift from Dr. Michael Grigg

and used in in vivo experiments. RHASubl were a kind gift from Dr. Vein Carruthers and

generated as described (Lagal et al. 2010). HFFs were maintained as previously described

(Rosowski et al. 2011). Lewis BMDMs were prepared as previously described (Cirelli et al.

2014). Cell Viability experiments and parasites per vacuole counts were performed as previously

described (Cirelli et al. 2014).

Western Blot

Western blots probing for IL-1p were performed on infected cell supernatants

concentrated using Amicon filters (3kD molecular weight cutoff) (Millipore, Billerica, MA).

Mutagenesis Screen

Intracellular wild-type RH parasites expressing GFP and Luciferase were treated with N-

ethyl-N-nitrosourea (ENU, 40uM), ethyl methanesulfonate (EMS, 1 OOuM) or dimethyl sulfoxide

(DMSO) for 4 hours. Parasites were washed three times with PBS, syringe lysed and allowed to

infect fresh HFFs. For selection, Lewis BMDMs were infected with parasite populations (MOI =

0.2 - 0.3) for two hours. Non-invading parasites were removed by washing cells with PBS three

times. Media was replaced with DMEM containing 30mg/ml dextran sulfate. At 24 hours post-

infection, extracellular parasites were removed by washing cells with PBS five times. Cells were

173

scrapped into fresh DMEM and overlaid onto fresh HFFs. Populations were selected for five to

nine rounds. Parasites were cloned via serial dilution.

Freshly lysed parasites were washed with 50ml PBS and filtered through 5pm syringe

filter (Millipore) to remove host cells. Parasite DNA was isolated using Qiagen DNeasy Blood &

Tissue Kit according to manufacturer's protocol. Parasite RNA was isolated from HFFs infected

for 48 hours using Qiagen RNeasy Mini Kit. Illumina sequencing was performed on Illumina

HiSeq 2000 or MiSeq. Reads were aligned using type I GT1 (v9.0) as reference genome.

Generation of parasite strains

Individual knockout of candidate genes was performed using CRISPR-Cas9. Sequences

targeting candidate genes were cloned into the pSS013 Cas9 vector (Sidik et al. 2014) . The

sequences are available in Supplementary Table 1. Plasmid containing gRNAs were co-

transfected with XhoI (New England Biolabs) -linearized pTKOatt (Rosowski et al. 2011) into

wild-type RH parasites at ratio 10:1. 24 hours post-transfection, populations were selected with

mycophenolic acid (50Rg/ml) and xanthine (50gg/ml) and cloned by limiting dilution. Knockout

was assessed by polymerase chain reaction (Supplementary Figure 1).

Complemented strains were generated by cloning gene with its putative promoter (-2000

bp upstream of start codon) with C-terminal hemagglutinin (HA)-tag sequence into pENTR

using TOPO cloning (Invitrogen) and then into pTKOatt using LR recombination (Invitrogen).

Prior to transfection, plasmids were linearized using a restriction enzyme with a unique

restriction site. Plasmid was co-transfected with plasmid containing dihydrofolate reductase

(DHFR) resistance cassette at ratio of 20:1. 24 hours post-transfection, populations were selected

174

with pyrimethamine (1 gM) and cloned by limiting dilution. Presence of tagged gene was

determined by immunofluorescent assay (IFA) and Western blot.

Immunofluorescent Microscopy

Cells were fixed with 100% ice cold methanol for 5 minutes and permeabilized with

0.2% TritonX-100. Colocalization studies were performed with anti-GRA7 or anti-ROPI and

anti-HA antibodies. Alexa Fluor 488 and 594 secondary antibodies were used, respectively as

previously described (Rosowski et al. 2011).

Plaque Assays

HFFs were grown to confluency in a 24 well plate. 100 parasites were added to each well

and incubated for 5 days at 37'C. The number of plaques was counted using a microscope.

Plaques were photographed using a digital camera (Coolsnap EZ; Roper Scientific) connected to

an inverted microscope (Eclipse Ti-S; Nikon) and plaque size was measured using NIS-Elements

software (Nikon).

Rat infection and rederivation

Tachyzoites were grown in HFFs and mechanically removed from host cells by passage

through a 27-gauge needle, followed by a 30- gauge needle. Parasites were washed three times

with PBS, quantified and diluted in PBS. Rats were infected using 27-gauge needle. After 30

days of infection, rats were sacrificed. Brains were harvested and homogenized in PBS by

passaging though 21-gauge needle. 1 / 1 0 th of brain suspension was added to confluent HFFs in T-

25 flasks in DMEM, supplemented with 1% heat-inactivated FBS, penicillin and streptomycin

175

and incubated at 37'C for 4 weeks. Parasite growth in vitro was usually observed around 2

weeks post-inoculation.

RNAseq

Brown Norway BMDMs were infected for 18 hours with parasites (MOI =1) in 6 well

plates. Plaque assays were performed at time of infection to determine viability and actual MOI

of each strain. Total RNA was isolated using Qiagen RNeasy Plus kit. RNA was prepared for

Illumina sequencing according to protocols. Sequencing was performed on Illumina HiSeq 2000.

Reads were mapped to the rat genome (RGSC3.4). To identify the pathways modulated by

GRA 18, GRA27 and/or GRA28, we focused on rat genes with FPKM >5 in at least one sample

and at least a two-fold difference in expression between any mutant and its complemented strain.

GENE-E was used to determine hierarchical clusters. PANTHER (pantherdb.org) was used to

investigate enrichment. REVIGO was used to visualize gene ontology enrichment.

176

Supplementary Figures

Primer Name Sequence CommentsTGGT1_236870_promF CACCCCAGACTTGGAATAGCGGTGAG Forward primer for

amplifying gene andpromoter

TGGTI_236870_HAR CTACTAAGCGTAATCTGGAACATCGT Reverse primer forATGGGTACCTGAGGAACGAGTTGTTT amplifying gene

TGGTI 236870 IF AAGTTGCAGCCAATCCTAACTGAATTG Forward oligo for gRNA #1TGGTI_236870_IR AAAACAATTCAGTTAGGATTGGCTGCA Reverse oligo for gRNA #1TGGTI_236870 2F AAGTTGATACGCCTTCTTTTGCGAAG Forward oligo for gRNA #2TGGT1_236870 2R AAAACTTCGCAAAAGAAGGCGTATCA Reverse oligo for gRNA #2TGGT1_236870_MutF GACACTCGTCGTTACCCGCAT Forward primer within gene

to determine KO

TGGTI_236870_MutR GAAATCCTTACGCCGGGAAGG Reverse primer within geneto determine KO

TGGT1_226380_overF CACCGGTACTAAACCAGACTGTGCCCG Forward primer foramplifying gene andpromoter

TGGTI_226380_HAR CTATCAAGCGTAATCTGGAACATCGTA Reverse primer forTGGGTAAGTCTGTTTCGGCTCCGCCTG amplifying gene with HA-

tag

TGGT1_226380_IF AAGTTGCAGCGACTGTTATATAAAGTG Forward oligo for gRNA #1TGGTI_226380 IR AAAACACTTTATATAACAGTCGCTGCA Reverse oligo for gRNA #1TGGT I236380 2F AAGTTGTAGCACAAGCATTAGTTGAG Forward oligo for gRNA #2

TGGT1_226380_2R AAAACTCAACTAATGCTTGTGCTACA Reverse oligo for gRNA #2TGGT1_226380 MutF ATGTATCCCCTGACTGTTTAC Forward primer within geneTGGT1_226380_MutR TCAAGTCTGTTTCGGCTCCGC Reverse primer within gene

to determine KO in type I

TGME49_226380_MutR TCAAGTCTGTTTCGGTTCCGC Reverse primer within geneto determine KO in type II

TGGTI_237015_overF CACCGCTCGAATCAAGGGTAGTTCAGG Forward primer forG amplifying gene and

promoter

TGGTI_237015_HAR CTATCAAGCGTAATCTGGAACATCGT Reverse primer forATGGGTATCCACTACCAGGAGCTCCTC amplifying geneC

TGGT1_237015_iF AAGTTGCTTGACTGGGCATTGCTCTGG Forward oligo for gRNA #1TGGT1 237015 IR AAAACCAGAGCAATGCCCAGTCAAGC Reverse oligo for gRNA #1TGGTI_237015 2F AAGTTGCTTGTTGAGGATATAGCACAG Forward oligo for gRNA #2TGGT1_237015 2R AAAACTGTGCTATATCCTCAACAAGCA Reverse oligo for gRNA #2TGGT1_237015_MutF ATGACCGTGCCCGTTCATCTG Forward primer within gene

to determine KO

177

Supplementary Table 1. Sequences of primers used in generating knockout andcomplemented strains. HA-tag is bolded.

178

TGGT1_237015_MutR CAGAGGCACCACGTCCATCAG Reverse primer within geneto determine KO

TGGT1_258580 IF AAGTTGAAATCGGGGATTTGTTCGTTG Forward oligo for gRNA #1

TGGT1 _258580 IR AAAACAACGAACAAATCCCCGATTTCA Reverse oligo for gRNA #1

TGGT1 258580 2F AAGTTGAAAGACCCTATTACCGTGATG Forward oligo for gRNA #2

TGGT1 258580 2R AAAACATCACGGTAATAGGGTCTTTCA Reverse oligo for gRNA #2

TGGT1_ 258580_MutF ATGGAGTTGGTGTTGTGCT Forward primer within geneto determine KO

TGGT1_ 258580_MutR GTTGCCCTGTGGTGGGATGTT Reverse primer within geneto determine KO

TGGT1_203040_IF AAGTTGACAGATTTCCTTCCCACTTCG Forward oligo for gRNA #1

TGGT1 203040 IR AAAACGAAGTGGGAAGGAAATCTGTC Reverse oligo for gRNA #1

TGGT1 203040 2F AAGTTGTGAAGCAGAGAGATATCTAA Forward oligo for gRNA #2

TGGT _203040 2R AAAACTTAGATATCTCTCTGCTTCACA Reverse oligo for gRNA #2

TGGT1_203040_MutF ATGCAACCTCTTCTCCCGCATTCTC Forward primer within geneto determine KO

TGGT1_203040_MutR AGCTCCTAAAAACGGACGATGAGCC Reverse primer within geneto determine KO

TGGT1 248260_IF AAGTTGTCTCCTTCAATTTAGTTCGTG Forward oligo for gRNA #1

TGGT1_248260_IR AAAACACGAACTAAATTGAAGGAGAC Reverse oligo for gRNA #1

TGGT1_248260_4F AAGTTGCATCACGGTTCCCACCAACG Forward oligo for gRNA #2

TGGT1 248260 4R AAAACGTTGGTGGGAACCGTGATGCA Reverse oligo for gRNA #2

TGGT1_248260_MutF CTTCTGGGTGGTCGAGTTCTT Forward primer within geneto determine KO

TGGT1_248260_MutR TCAATACGGACTTCCCGTGCT Reverse primer within geneto determine KO

\~( \~((~$0 $0

00 00 C>& \C~Q

~>(j$1S N

TGT1 203040(2.0kb)

Cjt,

TgGT1 22638~ OW 46 4(1. 1kb)

d, A00 0 0 C

TgGT1_258580(0.9kb) 0

TgGT1_237015(0.4kb)

)~\ ~&

00~ \NN/ $\/

b' -OT~ TC) C) C) Crv

g

XQ) XC)

TgGT1_248260 uso(0.7kb)

Supplementary Figureisolated from clones and

of interest. DNA quality

candidate gene or the B I

TgGT1_(1.5kb)

236870 OW O

1. PCR confirming knockout of candidate genes. Genomic DNA was

used as template. Knockout was determined by failure to amplify gene

was assessed by amplifying another Toxoplasma gene, either another

gene.

179

B1(1lO0bp)

Acknowledgements

This study was supported by the National Institutes of Health (RO 1 -AI080621) awarded

to JPJS. KMC was supported by National Institutes of Health (F31-AIL04170). MAH by a

Wellcome Trust-MIT postdoctoral fellowship.

180

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184

Chapter Five:

Conclusions and Future Directions

185

Summary

The work in this thesis demonstrated that the parasite Toxoplasma gondii activates the

NLRP1 and NLRP3 in the murine model. Deficiency of NLRP3, but not NLRP1, in bone

marrow-derived macrophages leads to a significant reduction in active IL-i IP secretion in vitro.

Inflammasome activation was not accompanied by pyroptosis. Mice deficient in NLRP1 or

NLRP3 were more susceptible to infection than wild-type mice. NLRP3 deficient mice had

significantly lower levels of circulating IL-18 than wild-type mice. IL-18 and IL-IR deficient

mice were also highly susceptible to infection, with significantly higher parasite loads. These

findings establish Toxoplasma as an activator of two murine inflammasomes, which play a role

in mouse resistance to parasite infection.

Additionally, we presented Toxoplasma as the second identified activator of the rat

NLRP1 inflammasome. Infection with the parasite leads to the secretion of active IL-i IP and IL-

18 from BMDMs from certain strains of rats. In contrast to our findings in murine macrophages,

infection of rat macrophages led to pyroptosis and the control of parasite replication. This

phenotype was abrogated when NLRP1 expression was reduced using RNAi.

To identify the parasite protein product(s) involved in rat NLRP1 activation, we

presented a forward genetic screen used to isolate parasites that fail to induce pyroptosis in rat

macrophages and are able to replicate. Infection with these parasites resulted in significantly less

active IL-i release. We identified three novel dense granule proteins, GRA18, GRA27 and

GRA28 as individually required for NLRP 1 inflammasome activation in vitro.

186

Discussion and Future Directions

The success of Toxoplasma gondii relies on its remarkable ability to reach a delicate

balance between immune activation and immune evasion. It must be sensed by the immune

system, where host mechanisms prevent excessive parasite replication, which could result in host

death before transmissible tissue cysts are formed. The parasite actively induces a strong immune

response. However, if the host mounts too strong of a response, infection will be completely

cleared. To prevent this unfavorable outcome, Toxoplasma has also evolved to evade these host

immune mechanisms. It is unlikely that Toxoplasma can establish an optimal balance between

immune activation and immune evasion in each of the different hosts it is capable of infecting,

resulting in differences in host susceptibility to toxoplasmosis. This balance is demonstrated by

inflammasome activation in mice. NLRP1 and NLRP3 activation by Toxoplasma leads to the

activation of IL- 18, which can lead to the production of IFN-y. Induction of this cytokine and its

downstream mechanisms leads to the control of the parasite and its conversion into the semi-

dormant bradyzoite in muscle tissues. In contrast, activation of the Lewis inflammasome by

Toxoplasma in rat macrophages leads to host cell death, preventing replication and resulting in

the complete clearance of the parasite.

Toxoplasma is capable of infecting all warm-blooded animals and must be equipped to

modulate the immune response of each host. As the parasite evolved, host immune pathways

evolved as well. Vertebrates often differ in their TLR and IRG repertoire. For example, while

TLR 1I and TLR12 play an important role in the sensing of Toxoplasma in mice, humans express

neither, leading to the need for additional mechanisms for parasite control. Additionally, the IRG

system utilized by many rodents to control Toxoplasma infection is not present in humans.

Substantial differences in host defense mechanisms have also been demonstrated. IRG proteins

187

are highly polymorphic between strains of mice, determining their susceptibility to Toxoplasma

(Lilue et al. 2013). Our work demonstrates the difference between host mechanisms as

Toxoplasma infection in Lewis BMDMs leads to rapid cell death, while infection of Brown

Norway, Sprague-Dawley or murine BMDMs does not.

How are the inflammasomes activated in the murine model?

We found a role for primarily NLRP3 and to a lesser extent, NLRP1 in the resistance of

mice to Toxoplasma. The mechanism of activation of NLRP3 is controversial. NLRP3 activation

requires a priming step, which upregulates the expression of both NLRP3 and pro-IL-i IP

(Bauernfeind et al. 2009). NLRP3 inflammasome activation can occur in response to both

external and endogenous stimuli. Extracellular ATP activates NLRP3 through the activation of

the purinergic receptor, P2X7R, leading to potassium efflux (Kahlenberg and Dubyak 2004;

Surprenant et al. 1996). Interestingly, Toxoplasma relies on potassium and calcium alterations as

a signal for parasite egress.

Regulation of NLRP3 includes the guanylate-binding protein 5 (GBP5) (Shenoy et al.

2012), which is recruited to the PVM and plays a role in parasite elimination (Winter et al.

2011). The mechanism through which GBP5 regulates NLRP3 activation is unclear although

several models have been proposed. GBPs may mediate in the lysis of the PV and allow for the

release of PAMPs, which are recognized by NLRP3 (Meunier et al. 2014). Another model

suggests that GBP5 can directly interact with NLRP3 and directly activates the inflammasome

(Shenoy et al. 2012). Recently, a third model has been proposed where PAMPs induce GBP5

oligomerization and this aids in NLRP3 oligomerization (Finethy et al. 2015).

188

Cellular stress, including changes in cell volume (Compan et al. 2012), an unfolded

protein response (UPR) (Menu et al. 2012; Kim et al. 2014), and reactive oxygen species (ROS)

(Heid et al. 2013) are able to activate NLRP3. As Toxoplasma extensively modifies the host cell,

it is possible the parasite indirectly activates the inflammasome by inducing cellular damage.

Some strains of Toxoplasma have been found to recruit the host mitochondria to the PV (Jones

and Hirsch 1972; Pernas et al. 2014). Perhaps the process of sequestering host mitochondria

damages the organelle, resulting in the release of ROS. Additionally, infection of mouse

macrophages results in respiratory burst (Wilson, Tsai, and Remington 1980).

We cannot rule out the possibility that Toxoplasma directly activates NLRP3 or

differentially activates NLRP3. Although the strains tested were able to induce active IL-i IP

secretion from LPS-primed mouse BMDMs with no significant differences (Chapter 2, Figure

1H), we did not perform a comprehensive survey of Toxoplasma strains. It is possible that

testing several more strains may allow us to identify a strain unable to activate the NLRP3

inflammasome. By performing a sexual cross between this strain with a strain that can activate

NLRP3, we can isolate a number of progeny that differ in phenotype. We can then conduct a

quantitative trait locus analysis and determine the genomic regions that correlate with the

phenotype.

How do GRA18, GRA27 and GRA28 activate the rat NLRP1 inflammasome?

Our genetic studies have demonstrated that GRA 18, GRA27 and GRA28 are required for

NLRPI activation in vitro. It is now of interest to determine how GRA18, GRA27 and GRA28

interact with each other, whether in a complex or within a pathway, and how they are able to

activate NLRP1. GRA18, GRA27 and GRA28 each have predicted transmembrane domains. We

189

hypothesize that these proteins reside within the parasitophorous vacuole membrane, facing into

the host cytoplasm.

We are interested in determining whether expression of one or more of these genes is

sufficient to activate NLRP1. Two approaches could be used to test this. The proteins can be

recombinantly expressed and purified from E. coli and transfected individually or in combination

into Lewis BMDMs using a liposomal transfection reagent, DOTAP, which will delivery the

proteins into the host cytosol. This method has successfully been used to identify flagellin as an

activator of NLCR4 in mouse macrophages (Franchi et al. 2006). There are several potential

limitations of this method. Purified proteins may contain bacterial components that themselves

can activate immune pathways, including the inflammasomes, in rat macrophages. Transfecting a

purified GRA that is unrelated to GRA18, GRA27 or GRA28 can serve as a control for possible

contamination. Additionally, this method may not be feasible if the proteins require post-

translational modifications for NLRP1 activation. A more reasonable method is ectopically

expressing the protein(s) using an inducible system. However, this approach may not work if

GRA18, GRA27 or GRA28 require processing by other Toxoplasma proteins for proper

function. Attempts at generating a Lewis macrophage cell line that behaves like primary

macrophages has not be fruitful. We have created several independent immortalized macrophage

cell lines using J2 virus, which uses v-raf and v-myc to induce cell proliferation (Blasi et al.

1989). Immortalized macrophages did not undergo pyroptosis when infected with Toxoplasma

and allowed for parasite replication. Additionally, these cells do not secrete IL-i I3 when primed

with LPS and infected or treated with the NLRP3 agonist, nigericin. We found Nlrpi, Caspi and

Caspli expression was reduced dramatically in these cell lines compared to BMDMs as

190

measured by qPCR, which may explain why the inflammasomes are not properly activated in

these cells.

As inflammasome sensors are expressed in the host cytoplasm, it is most likely a parasite

protein that is trafficked out of the parasitophorous vacuole that is recognized by NLRPI. We did

not detect export of GRA18, GRA27 or GRA28 into the host cytosol as measured by

immunofluorescent microscopy. To determine the precise location of these proteins, we plan to

perform cell fractionation studies of infected cells using ultracentrifugation and determine if

these proteins are associated with the PVM (Neudeck et al. 2002; Sibley et al. 1995). While this

method has been successful for identifying the location of proteins within the PVM, a more

precise and sensitive method utilizes immunoelectron microscopy. This approach will allow us

to study the subcellular localization of the proteins in situ.

As GRA 18, GRA27 and GRA28 potentially interact with several other parasite proteins

and host proteins, it will be interesting to determine these unknown interactors. We have

performed immunoprecipitations of GRA27 and GRA28 from infected HFFs, but have not been

able to IP GRA18. Although immunoprecipitations using whole infected cells allowed for the

pull down of nearly all of GRA27 and GRA28, this experimental setting may yield misleading

results if submitted for mass spectrometry. As dense granule proteins are continually translated

during replication within the cell, a fraction of our proteins of interest (POIs) that we isolate are

located within the dense granule organelle. Within this organelle are a wealth of GRAs, which do

not normally interact with our POIs when secreted from the parasite. By performing

immunoprecipitations of POIs remaining in the dense granule, we may detect other GRAs that

are not biologically relevant. Therefore, it is worth performing a step prior to

immunoprecipitation to remove intact secretory organelles. Another method involves expressing

191

GRA18, GRA27 or GRA28 recombinantly and immobilizing these proteins onto a matrix. We

can isolate interacting proteins by allowing whole host cell or parasite lysates to incubate with

POls and identify the proteins via mass spectrometry.

As it is unlikely that GRA18, GRA27 or GRA28 are directly recognized by NLRP1, it is

still of interest to identify the direct activator. Using the mutagenesis screen we described in

Chapter 4, we isolated seven independent clones. We identified the causative mutations in four

of these isolates. The three remaining clones have been sequenced and do not have mutations in

Gra18, Gra27 or Gra28 nor do they share mutated genes with each other (Chapter 4, Table 1).

By focusing on genes with predicted signal peptides, we can narrow this list to four candidate

genes, TgGTJ_309160 (Mutant #5), TgGTJ_203230 (Mutant #6), TgGTJ_408760 (Mutant #7)

and TgGTJ_410880 (Mutant #7). Using CRISPR/Cas9, we can knock these genes out with

relative ease. One or more of these genes may not play a role in activation of the Lewis

inflammasome. It is possible the causative mutations in these clones are within genes that encode

for proteins that do not contain predicted signal peptides. We can then expand our candidate list

to include all genes expressed by all Toxoplasma strains. We potentially can identify three more

Toxoplasma genes required for NLRP1 activation. By expanding this set of proteins, we are

more likely to find the activator or elucidate the pathway leading to the recognition of the

activator.

Although we have focused on genetic approaches to identify the NLRP1 activator, a

biochemical approach may provide a more direct method. BiolD utilizes a promiscuous biotin

ligase, BirA*. When fused to a POI, BirA nonspecifically biotinylates proteins within close

proximity (Roux, Kim, and Burke 2013; Roux et al. 2012). Modified proteins can be isolated

using streptavidin-coupled beads. A major advantage of this approach is this method will allow

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for the identification of transient interactors. Additionally, BirA*-fused proteins can be

transfected in a cell line, such as HEK293 cells, to generate a stable cell line. These cells will not

undergo pyroptosis as human cell lines engineered to express NLRP1 and caspase-1 do not die

when infected with Toxoplasma (Chapter 3, Figure S8). We have fused BirA* to Lewis NLRP1,

but have failed to construct a stable HEK293 line expressing this protein. Once a cell line

expressing the construct is cloned, cells can be infected with Toxoplasma and isolated modified

proteins will be submitted for mass spectrometry. As we found no difference in IL-i I3 secretion

between murine macrophages infected with wild-type and strains deficient in GRA 18, GRA27 or

GRA28, this method may also be used to identify parasite proteins that may interact with murine

NLRPL.

Why are GRA18, GRA27 and GRA28 conserved among strains?

A survey of many Toxoplasma strains that represent worldwide diversity demonstrated

that all tested strains of the parasite were able to induce pyroptosis in Lewis BMDMs (Chapter

3, Figure 3). This finding demonstrates a well-conserved phenomenon. As induction of

pyroptosis leads to the control of parasite replication, it is likely the Lewis rat and other rat

strains that are resistant to Toxoplasma evolved to recognize features of the parasite that are

widely conserved. In addition, a close relative of Toxoplasma, Neospora is able to induce

pyroptosis in the same cells, suggesting that some rats have adapted to identify conserved

parasite features (Chapter 3 - Addendum, Figure 1). As GRA18, GRA27 and GRA28 share

homology to proteins in Hammondia hammondi, it will be interesting to test the ability of this

species to induce pyroptosis in rats.

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New parasite strains are a result of sexual recombination of parental strains in felines. As

the parasite can infect a wealth of intermediate hosts with different host mechanisms, selection of

a strain is heavily determined by the host immune system. Polymorphisms in parasite effectors

have been well documented to determine virulence in laboratory mice. Conservation of GRA18,

GRA27 and GRA28 suggests a selective pressure on these proteins and indicate that they may

play an important role in the success and transmission of the parasite. The genes are not essential

in tachyzoites as we were able to generate strains deficient in each gene. A preliminary

experiment in susceptible rats suggests these genes may play a role in establishment of a chronic

infection, but future experiments must be conducted in both mouse and rats.

We performed preliminary RNAseq experiments on Brown Norway rat macrophages

infected with strains deficient in GRA8, GRA27 or GRA28 and their complemented control

strains. Expression of several type I interferon-stimulated genes, including Ifi27, IsgJ2(b) and

Glp2 were reduced in cells infected with strains deficient in GRA27 or GRA28 compared to

control strains (Chapter 4, Table 3). While type I IFN is an established inducer of these genes,

only two Toxoplasma strains, BOF and COUGAR, have been identified to induce type I IFN

(Melo et al. 2013). IFN-independent pathways can induce interferon-stimulated genes. (Noyce et

al. 2011). Toxoplasma may activate one of these alternative pathways. In addition to the type I

IFN-inducible genes, we found genes involved in leukocyte chemotaxis significantly enriched

(p-value = 1.2 x 10-6). These genes included Cxcl, Cxcl2, Cxcl10 and Cxcl]1. This suggests

GRA27 and GRA28 play a role in activating an immune response other than the inflammasomes.

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Why are rats so resistant to Toxoplasma?

While we have focused on the extraordinary resistance of the Lewis rat to Toxoplasma,

we are also interested in determining why all rat strains are resistant to the parasite. Through the

use of neutralizing antibodies, IFN-y was determined to be important in the control of the

parasite in susceptible rats (Sergent et al. 2005). We have performed in vitro experiments testing

the role of IFN-y in control of the parasite in nonimmune cells. Preliminary experiments

confirmed that IFN-y plays a role, as the parasite formed fewer and smaller plaques in rat

fibroblast monolayers prestimulated with IFN-y than those untreated.

Polymorphisms exist in IRG proteins between mice strains. The most studied laboratory

mouse is relatively susceptible, with a single type I parasite able to kill a mouse within a week.

This virulence is determined by the ability of parasite effectors, ROP 18 and ROP5 to counter the

host IRG system. The IRG system in wild mice is polymorphic and in some strains, not

susceptible to the actions of ROP18 and ROP5, allowing these strains to survive infection with

Toxoplasma (Lilue et al. 2013). Rat IRG proteins are similar to the IRGs in these resistant wild

mice, suggesting that the IRGs may play a major role in rat resistance. We are generating rat

fibroblasts that do not express IRGM, an important regulator of IRG oligomerization on the

PVM, and ATG5, an ubiquitin ligase necessary for autophagy and important for IRG formation

(Zhao et al. 2008; Ohshima et al. 2014). Using these cell lines, we plan to determine if the IRGs

play a role in the control of Toxoplasma in rat non-immune cells.

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.. ..... ...

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