waltz or rave? how polymorphic effectors influence ... · waltz or rave? how polymorphic effectors...

Waltz or Rave? How polymorphic effectors influence Toxoplasma’s dance with its host

John Boothroyd Stanford University

at Zoobiquity April. 5, 2014

Or Rave…

J.P.J. Saeij, J.P. Boyle et al. Infection & Immunity 2005

Waltz

500 Toxoplasma strain S22 inoculated


What is the molecular basis for these differences in virulence?

Genetic crosses allow the mapping of Toxoplasma genes responsible for a phenotype

Gametes

Oocysts

carnivorism

Clone, map and phenotype recombinants.

>109 progeny

ME49 (Type II)

CEP (Type III)

F1 progeny of a cross between Types II and III show virulence is a quantitative trait

F1 progeny from Pfefferkorn; mapped by Sibley et al., Genetics, 1992 and Khan et al., NAR, 2005

< 10 E7 S23~ 100 A5 STH1 STG2 STF3 STC8 CL11 CL16 CL19~1000 C12 H6 STH10 S30~10,000 S2910,000-100,000 STG4 STG10 CL17 CL12 STD10>100,000 STD3 S28 CL13 S21 S25 S27 STH5 S2T CL18 S1T

B4 STE1 STD2 STC7 STE7 STE10 STH11 S22 S26 CL15

Recombinant F1 progeny from II x III cross: ~LD50:

Grigg et al Science 2001 Saeij et al. Science 2006

(II)

(III)

Or Rave

J.P.J. Saeij, J.P. Boyle et al. Infection & Immunity 2005 S22 S23

II III

F1-S23 50 parasites

Waltz

F1-S22 500 parasites 500 Toxoplasma strain S22 inoculated


Toxoplasma is an obligate intracellular parasite that injects rhoptry proteins (ROPs) early in invasion!

B. Nicholls (1983); S. Hakansson & D. Sibley (2001)

Kimata and Tanabe (1987)

Anti-ROP9

Discharging rhoptries

1 µm

Dense granules

Dance Step 1: ROP16 is a polymorphic tyrosine kinase that directly phosphorylates host STAT3/6

Susan Coller, Ching Ong, Jeroen Saeij, Jon Boyle, and Anjali Shastri

(STAT = Signal Transducer and Activator of Transcription)

Phosphorylation of STAT3/6 20 hours post-infection differs between the predominant Toxoplasma Types!

Saeij et al. Nature 2007

Saeij et al., Nature, 2007

RH (Type I)

ROP16 is a JAK2 mimic responsible for the (VERY) rapid ��tyrosine phosphorylation of STATs

HFFs, 1 min p.i.

RHΔrop16

phase DAPI pSTAT6 (Y705) ROP 2/3/4 ROP 2/3/4

Yamamoto et al., JEM, 2009; Ong et al., JBC, 2010 Ong et al., J. Biol. Chem., 2010

Jeroen Saeij, Jon Boyle and Michael Reese

+Sibley lab +Howard lab

Dance steps 2 and 3: ROP5 and ROP18 are polymorphic ROPs that collaborate to block a key

effect of Interferon-gamma

ROP18 is a polymorphic ser/thr kinase that localizes to the parasitophorous vacuole membrane (PVM)

• Type I/II are ROP18+; Type III are ROP18-

Type I (ROP18+) Type III (ROP18-)

pvm pvm

n n

J-F Dubremetz

ROP18 phosphorylates IRGs at the parasitophorous vacuole membrane (PVM)

• IRGs are small GTPases induced by IFNγ that attack the PVM

Steinfeldt et al., 2010; Fentress et al., 2010

I

IRGs attack PVM No IRGs at PVM No IRGs at PVM (ROP18+) (ROP18-)

PVM PVM

Steinfeldt et al., 2010; Fentress et al., 2010

I

IRGs attack PVM No IRGs at PVM No IRGs at PVM (ROP18+) (ROP18-)

PVM PVM

ROP18 phosphorylates IRGs at the parasitophorous vacuole membrane (PVM)

• IRGs are small GTPases induced by IFNγ that attack the PVM

ROP5

Unbound IRG

T102/T108 (phosphosites)

ROP5 is a pseudokinase that binds and alters Irga6 conformation, exposing target phospho-sites of ROP18

M. L. Reese, unpublished

T102/T108 (phosphosites)


Bound IRG

ROP5

ROP5 is a pseudokinase that binds and alters Irga6 conformation, exposing target phospho-sites of ROP18

ROP5

IRG

Strain-specific polymorphic residues in green Isoform-specific polymorphic residues in yellow

ROP5 pseudoactive site

ROP5 polymorphisms cluster at IRG-binding site


Lena Pernas

Anjali Shastri

Moritz Treeck

Polymorphonuclear cell isolated from mouse peritoneum 6 dpi with Toxoplasma - RH Polymorphonuclear cell isolated from mouse peritoneum 6 dpi with Toxoplasma

� µ � �

Toxo

M N

N

N

M

M

M

M

Dance step 4: Polymorphic MAF1 mediates strain-specific association of Toxoplasma with host mitochondria

Toxoplasma MitoTracker

Human foreskin fibroblasts

Association of Toxoplasma-RH and host mitochondria occurs in all cells tested.

Mitotracker

BUT, not all strains of Toxoplasma show mitochondrial association!

Type II

Type III

Mitotracker

Mitotracker

Type I

Pernas et al., PLoS Biology, in press

MAF1 (Mitochondrial Association Factor 1) is necessary for host mitochondrial association (HMA):

Knock-out of MAF1 in a Type I strain converts to HMA-

5µm

Merge

µ

α� � � � � � � � � � � � � � � � � � �RFP

RH Δmaf1 RFP+ RH wt

Phase


MAF1 acts alone: HA-tagged MAF1 expressed in mouse embryonic fibroblasts localizes to the mitochondria

*

*

*

* * *

*

* *

αTom20 αHA � � � � � � µ � �


host plasma membrane!

Stat ROP16

pStat [active]

pStat

host nucleus!

rhoptries dense granules

Summary of the Dance Moves

And: ROP38 – down-regulates MAPK; Peixoto et al. 2010 GRA15 – activates NF-kB; Rosowski et al., 2011 GRA24 – activates p38 MAPK; Braun et al., 2013

ROP16 ROP5 ROP18

parasitophorous!vacuole!

!

IRG

pIRG [inactive] pIRG

ROP18

ROP5

MAF1

Modulation of NF-kB, IRF3, IRF7 signaling?

MAF1

What is the reason for these differences in virulence?

Reminder of the life cycle

carnivorism

grazing

Intermediate Hosts

Bradyzoites (tissue cysts)

Tachyzoites

ASEXUAL

Definitive Host

Gametes

Oocysts

SEXUAL

Hypothesis: Do strain-specific differences exist to optimize infection of different strains in different host species??

STAT6 (murine/human)

ROP16 (Type I/III)

ROP16 (Type II)

STATx (species X)

So, repeat these studies in avian cells – different from results in mammals?

Ong et al., PLoS ONE, 2011

Maybe it’s not the host species but the host ecology that selects for different ROP16 alleles?

STAT6 (murine)

ROP16 (Type I/III)

Inflammatory response needs to be counter-balanced by upping Th2 (via STAT6 activation)?

ROP16 (Type II)

STAT6 (murine)

Th2 already dominates because of worms so not good to push further in that direction?

P

What is the clinical relevance of these strain-specific differences in

virulence?

Strain-type may explain some differences in disease-outcome in humans

J. Inf. Dis. 2001

Europe

Brazil

Gilbert et al. PLoS NTD 2008

years

Frac

tion

free

of e

ye le

sion

s

Gilbert et al., PLoS NTD, 2008

1. Strains differ dramatically in the character of their dance with the host.

S22 S23

3. Host range is likely the driver of these strain-specific differences but maybe it’s the overall “ecosystem” of the host, not host species, that is the variable

Summary

4. Strain-specific differences appear to matter in human infections, as well.

Brazil

Europe

years

Frac

tion

free

of e

ye le

sion

s

2. Strain-specific differences in virulence are a result of huge differences in the effectors introduced by different strains.

Type I Type II

HMA+ HMA-

In Summary:

Colleagues and Collaborators:

Original crosses: Elmer Pfefferkorn – Dartmouth!Mapping: David Sibley – Washington Univ.!

Genome Sequencing: Sequencing Consortium and the ToxoDB team!ROP5/Irga6: Jonathan Howard, Tobias Steinfeldt and Martin Fleckenstein,

Cologne!MAF1: Jon Boyle and Yaw Adomako-Ankomah – Pittsburgh!

Michele Tonkin and Marty Boulanger – Victoria! !

THANKS!

Moritz Treeck Lena Pernas Michael Reese Anjali Shastri

1. Strains differ dramatically in the character of their dance with the host.

S22 S23

3. Host range is likely the driver of these strain-specific differences but maybe it’s the overall “ecosystem” of the host, not host species, that is the variable

Summary

4. Strain-specific differences appear to matter in human infections, as well.

Brazil

Europe

years

Frac

tion

free

of e

ye le

sion

s

2. Strain-specific differences in virulence are a result of huge differences in the effectors introduced by different strains.

Type I Type II

HMA+ HMA-

In Summary:

waltz or rave? how polymorphic effectors influence ... · waltz or rave? how polymorphic effectors...

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