mim phd program - eth z · welcome to the 8th student retreat of the mim phd program dear...

MIM PhD Program Microbiology & Immunology

8th MIM PhD Retreat 27th ‐ 29th of August 2015

Fiescheralp, Aletsch Arena Switzerland

Sponsored by SUK‐Programm "Doktoratsprogramme" (ETH Zurich & UZH)

2

Contents

Welcome to the 8th Student Retreat of the MIM PhD Program .......................... 3

Program overview ................................................................................................ 4

Detailed program ................................................................................................. 5

Guest speakers ..................................................................................................... 7

Student talk abstracts .......................................................................................... 9

Poster abstracts ..................................................................................................18

Poster session I ...................................................................................................57

Poster session II ..................................................................................................58

Participant details ..............................................................................................60


3

Welcome to the 8th Student Retreat of the MIM PhD Program

Dear Participants,

We hope you will enjoy this year`s MIM retreat. The Fiescheralp is a great place to escape from the daily lab routine and get inspired by all the research that is happening within the Graduate School program. The aim of this retreat is manifold. One important aspect is to give you a taste of what other students work on. We want to boost the interaction between different Labs. Therefore, we encourage you to get out most of the poster session and the student talks. For a wider perspective we invited several excellent guest speakers who will talk about the life as a scientist and beyond. The time of lone warriors in science has passed. That is why we see this retreat also as a meeting to socialize with fellow researchers, start new collaborations and friendships. We are looking forward to the following days with great scientific discussion in a relaxed atmosphere. Contact information

In case of emergency or if something needs to be clarified, please feel free to contact any of

us via e‐mail or phone:

Name Mobile phone No. E‐mail

Isabel Barnstorf 0788807873 [email protected]

Julia Rühl 0766101923 [email protected]

Leoni Swart 0763328690 [email protected]

Bernhard Steiner 0763034732 [email protected]

Dagmara Lewandowska 0768244566 [email protected]

Rebecca Higgins 0787380870 [email protected]

Mario Hupfeld 0787367171 [email protected]

Location

Alpenlodge Kühboden

Familie Rickhoff‐Florian

Fiescheralp 1

3984 Fiesch

Phone: +41 (0)27 970 12 20

Homepage: http://www.kuehboden‐fiescheralp.ch/


4

Program overview

Time/date Thursday 27th Friday 28th Saturday 29th

7:00 Breakfast

7:30

Departure from Zurich HB

8:00 8:25 cable car up

8:30 Student session 2

9:00 Dr. Christoph Burkhart Breakfast

9:30

10:00 Coffee break

Hike & Lunch

10:30

Student session 3 11:00 Arrival & welcome

11:30

12:00 Lunch Lunch

12:30

13:00 13:25 cable car up Student session 4

13:30 Student session 1

Workshops: Dr. Urs Ziegler 14:00 Dr. Steve Pascolo

13:56 departure from Fiesch

14:30

15:00 Coffee break Coffee break

15:15

Dr. Heike Nowag 15:30 Introduction into glaciers

16:00 16:25 cable car down


17:00 Dinner Dinner

17:30


Socializing time 18:30

Poster session I 19:00


20:00

Poster session II

Party

20:30

21:00


22:00



5

Detailed program

Thursday, 27th of August

7:30 Departure Zurich HB

11:00 – 12:00 Arrival & welcome

12:00 – 13:00 Lunch

13:25 Going up with cable car

***************************************** Lecture session 1 *******************************************

Chair: Julia Rühl

13:45 – 14:05 Student talk 1: Dagmara Lewandowska

14:05 – 15:05 Guest Lecture I: Dr. Steve Pascolo, USZ, Miescher Pharma GmbH

15:05 – 15:15 Coffee break

15:15 – 16:15 Guest speaker II: MIM Alumni Dr. Heike Nowag, Consulting PWC

16:25 Going down with cable car

17:00 – 18:00 Dinner


18:30 – 20:00 Poster session 1 (A‐L)

20:00 – 21:30 Poster session 2 (M‐Z)


Friday, 28th of August

7:00 – 8:00 Breakfast


***************************************** Lecture session 2 *****************************************

Chair: Dagmara Lewandowska

8:30 – 8:50 Student talk 2: Weronika Barcik

8:50 – 9:10 Student talk 3: Franziska Schönherr

9:10 – 10:10 Guest Lecture III: Dr. Christoph Burkhart, Novartis Institutes for BioMedical

Research, Department Autoimmunity, Transplantation and Inflammation


6


***************************************** Lecture session 3 *******************************************

Chair: Rebecca Higgins

10:40 – 11:00 Student talk 4: Emanuel Stiegeler

11:00 – 11:20 Student talk 5: Michel Crameri

11:20 – 11:40 Student talk 6: Isabel Barnstorf

11:40 – 12:00 Student talk 7: Monica Loi

12:00 – 13 00 Lunch

***************************************** Lecture session 4 *******************************************

Chair: Mario Hupfeld

13:00 – 13:20 Student talk 8: Corinna Landig

13:20 – 13:40 Student talk 9: Bernhard Steiner

13:45 – 15:00 Workshops: Dr. Urs Ziegler, Center for Microscopy and Image Analysis



17:00 – 18:00 Dinner

18:00 – 19:30 Socializing/free time/rest


20:00 – 22:00 Party


22:00 ‐ ??? Party goes on in the restaurant

Saturday, 29th of August

9:00 – 10:00 Breakfast

10:00 – 12:30 Hike

13:56 Train back to Zurich

17:00 Arrival at Zurich HB


7

Guest speakers

Steve Pascolo, PhD

Group leader

Department of Dermatology, University Hospital of Zurich, Zurich, Switzerland Phone: +41 44 6342876 [email protected] Performing research leads eventually to inventions or discoveries that can be turned into patents.

Those can be licensed to companies or used as a cornerstone to create a biotechnology company

stemming from University: a spin‐off. By founding a company you have the advantage of creating your

own carrier opportunities, keeping in touch with university research and making links to industry. As

examples of possible paths, I will present the history of two different models of spin‐off that I (co‐)

created: CureVac GmbH in 2000 and Miescher Pharma GmbH in 2008. Thereby, I will introduce the

basic requirements and mechanisms that may help you setting your own company based on your (or

other’s) academic research results.

Heike Nowag, PhD Consultant ‐ Life Sciences & Pharmaceuticals

PricewaterhouseCoopers AG

Birchstrasse 160 | Postfach | CH‐8050 Zürich

http://www.pwc.ch

Phone: +41 79 574 5343

[email protected]

I completed my PhD at the University of Zurich in the field of Immunology in August last year, and was

faced with the decision on what to do next. I decided to leave the world of academia and took a

consulting position with PwC. In my presentation, I want to talk a little about the firm and what it is

like to work as a consultant in the field of Life Sciences & Pharmaceuticals. I would also like to share

my experience on making the transition to industry and useful lessons I have learned along the way.


8

Dr. Christoph Burkhart, PhD

Director

Department of Autoimmunity, Transplantation and Inflammation

Novartis Institutes for BioMedical Research, Basel, Switzerland

Phone: +41 79 7987523

[email protected]

The presentation will contain a time‐lapse view of my academic and professional career followed by

an introduction to my current workplace, the Novartis Institutes for BioMedical Research (NIBR). NIBR

is a global research organization with about 6,000 scientists dedicated to the discovery of truly

innovative drugs. It focusses on molecular pathways shared by various diseases and integrates clinical

insights with mechanistic understanding of pathological processes.

Drug discovery and drug development at Novartis will be exemplified by the journey of a compound,

the anti‐IL‐17‐specificmonocolobal antibody Secukinumab, from preclinical testing to Proof‐of‐

Concept and beyond. Finally, the possibilities of careers at Novartis through the NIBR Postdoctoral

Program will be described.

Urs Ziegler, PhD

Head of Center for Microscopy and Image Analysis, UZH

Center for Microscopy and Image Analysis, University of Zurich

Winterthurerstrasse 190, CH‐8057 Zürich

Phone: +41 44 635 53 55

[email protected]

Taking a deep look: modern microscopy technologies

Microscopy is widely used in life science. Progress in technology, but also in biology, keeps widening its scope and applications. The very basic theoretical background of light‐ and electron microscopy will be discussed. State of the art and upcoming imaging concepts in light and electron microscopy are summarized. Specifically, the focus will be on confocal laserscanning, light sheet and multiphoton microscopy but also transmission and scanning electron microscopy. All techniques will be discussed with a view on most widely used model organisms or samples. Preparation strategies for samples will be mentioned where appropriate.


9

Student talk abstracts

Microbiota‐Derived Histamine ‐ Relevance to Mucosal Immune Homeostasis

Weronika Barcik, Remo Frei, Liam O`Mahony

University of Zurich, Swiss Institute of Allergy and Asthma Research, Obere Strasse 22, 7270, Davos

Platz, Switzerland

[email protected]

Histamine is an important immunomodulator. It can influence protective or pro‐inflammatory immune

responses. In addition to host cells, histamine is also secreted by many different bacterial strains. In

this study, the aim is to determine the biological role, pathological significance and therapeutic

potential of bacterial‐derived histamine.

After PCR screening fecal samples from 160 patients and healthy volunteers we observed that the

bacterial HDC gene copy number was significantly higher in asthma patients compared to healthy

volunteers. Moreover atopic asthma patients had significantly increased bacterial HDC gene copy

number compared to non‐atopic patients. In order to identify the bacteria, which secrete histamine,

fecal samples were cultured on TSA plates supplemented with histidine. HDC gene expression by

isolated microbes was confirmed using RT‐PCR and histamine secretion into culture supernatants was

confirmed by ELISA. 16s sequencing of histamine secreting microbes is currently underway. Finally,

invariant natural killer T (iNKT) cells are important for appropriate mucosal immune responses. We

have shown that histamine modulates iNKT cell cytokine secretion and proliferation, raising the

possibility that bacterial‐derived histamine may influence iNKT cell activity at mucosal sites.

In conclusion, we have confirmed that bacteria present within the gut microbiome can secrete

histamine. Bacterial‐derived histamine may have clinical relevance as increased levels of these bacteria

are present within the microbiome of atopic asthma patients.


10

Impact of persistent viral infections on immune responsiveness in mice

Isabel Barnstorf, Annette Oxenius Institute of Microbiology, Department of Biology, ETH Zurich, Zurich, Switzerland [email protected] Immune responsiveness of a host towards microbial challenges or vaccines depends on the various

constituents of the immune system which are continuously modulated for instance by

the previous infection/vaccine history of an individual, the constant encounter with commensal

microorganisms at mucosal surfaces (the "microbiome") and the exposure of the immune system to

persistent viral infections (the "virome"). Persistent viral infections are wide‐spread in the human

population with estimated 8‐12 persistent viral infections per individual. Yet, in contrast to the

"microbiome", there is currently limited knowledge on how these persistent infections impinge on

immune homeostasis or immune responsiveness.

The aim of this project is to address the influence of two well‐defined persistent viral infections in the

mouse, Lymphocytic Choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV) on the long‐

term composition, phenotype and function of diverse innate and adaptive immune cells and how such

alterations affect vaccine efficacy, susceptibility to infection by heterologous pathogens,

predisposition to autoimmunity and immunological ageing.


11

Influenza A virus NS1 inhibits interferon‐mediated signaling independently of its block of general host gene expression

Michel Crameri and Jovan Pavlovic Institute of Medical Virology, University of Zurich, Winterthurerstrasse 190, CH‐8057 Zürich, Switzerland [email protected]

Type I interferons (IFNs) act as the first line of defense against viral infections. Upon secretion and

receptor binding, they initiate a signaling cascade that eventually leads to the establishment of an

antiviral state owing to the production of IFN‐induced effector proteins. The type I IFN signaling

cascade involves phosphorylation of signal transducer and activator of transcription 1 (STAT1) and

STAT2. Phosphorylated STATs heterodimerize and translocate to the nucleus, where they activate

transcription of IFN‐responsive genes. Previous studies have shown that influenza A virus (IAV)

infection causes disruption of IFN‐mediated signaling. Using a luciferase‐based reporter assay, we

found that overexpression of avian IAV non‐structural protein 1 (NS1) causes a dramatic reduction of

IFN‐induced gene expression. However, STAT1 phosphorylation was not affected. Instead,

immunofluorescence data demonstrated that NS1 interferes with IFN‐induced nuclear translocation

of STAT proteins. We then assessed whether NS1 proteins from multiple IAV strains differ in their

ability to suppress the signaling events in response to IFN. Intriguingly, most human and avian NS1

proteins which failed to inhibit general host gene expression remained effective IFN signaling

antagonists. In addition, mutation of NS1 residues essential for interaction with CPSF30 and

subsequent block in host mRNA maturation restored general gene expression, while still interfering

with IFN‐mediated signaling. Conversely, disruption of a conserved putative protein‐protein

interaction motif partially restored IFN signaling reporter activity. Taken together, we show that IAVs

have evolved multiple strategies to inhibit IFN‐mediated signaling, relying on both general and specific

suppression of host gene expression.


12

The human‐specific pathogen Neisseria gonorrhoeae engages innate immunoregulatory Siglec receptors in a species‐specific manner

Corinna Landig1,2, Jerry Fong2, Sarika Agarwal3, Flavio Schwarz2, Markus Aebi1, Victor Nizet2, Sanjay Ram3 and Ajit Varki2 1Institute of Microbiology, Department of Biology, ETH Zurich, 8093 Zurich, Switzerland; 2Glycobiology Research and Training Center, Departments of Medicine, Pediatrics and Cellular and Molecular Medicine, UC San Diego, La Jolla, CA 92093, USA; 3Department of Medicine, University of Massachusetts Medical School, Worcestor, MA 01605, USA [email protected] The human‐specific pathogen Neisseria gonorrhoeae causes the sexually transmitted disease

gonorrhea, which is a major global health burden and causes serious sequelae like infertility, especially

in women. It has been predicted that the recent vast increase in antibiotic‐resistant gonococcal strains

will lead to an upcoming era of untreatable gonorrhea. The pathogen has evolved different

mechanisms to evade the host immune system. One mechanism is the sialyation of its surface

lipoligosaccharide, which is known to generate resistance to the alternative pathway of complement,

as well as to mask underlying antigenic epitopes. The CD33‐related sialicacid binding immunoglobulin

like lectins (CD33rSiglecs) on innate immune cells recognize sialic acid bearing glycans, thus inducing

either anti‐inflammatory or proinflammatory responses. Based on precedents with other pathogens,

it is likely that anti‐inflammatory Siglecs are targeted by gonococci to exploit the host immune system,

and that pro‐inflammatory Siglecs represent a host evolutionary response to this exploitation.

We now show for the first time that N. gonorrhoeae can engage multiple human CD33rSiglecs that it

might encounter in the genitourinary tract, and that it preferentially bind human Siglecs over the

chimpanzee orthologs. These results can contribute to understanding of the mechanisms utilized by

N. gonorrhoeae to evade the host immune system and potentially to the development of novel

therapeutics to treat gonorrhea.


13

Resolution of undefined aetiology of respiratory infections in lung transplant patients with unbiased metagenomic sequencing

D. Lewandowska1, B. Ruehe1*, P. Schreiber2*, O. Zagordi1*, F. D. Geissberger1, M. Schuurmans3, A. Zbinden1, J. Böni1, C. Benden3, N. Müller2#, A. Trkola1#, M. Huber1#

1 Institute of Medical Virology, University of Zurich, Zurich, Switzerland; 2 University Hospital Zurich, Division of Infectious Diseases, Zurich, Switzerland; 3 University Hospital Zurich, Division of Pulmonary Medicine, Zurich, Switzerland; * and # contributed equally

[email protected]

Lung transplanted patients are a vulnerable group of immunocompromised individuals prone to

respiratory infections, necessitating a tight monitoring of viral infections post‐transplantation to

prevent respiratory complications and allograft dysfunction. Despite routinely testing for the most

common respiratory viruses, a considerable fraction of infections remains with unknown aetiology.

We re‐analysed samples collected from symptomatic lung transplant patients for which no viral or

microbial aetiology could be found. We used metagenomic approach to identify potential pathogenic

viruses responsible for the respiratory symptoms. For metagenomic sequencing, virus particles were

enriched from respiratory swabs, total nucleic acids extracted and randomly amplified. Sequencing

libraries were prepared and sequenced on MiSeq, Illumina. Quality filtered reads were cleaned from

non‐viral reads by an in‐house bioinformatics pipeline and blasted against a database containing

approximately 40,000 viral genomes. The metagenomic approach identified 3 cases of Rhinovirus A,

Rhinovirus B and Coronavirus HKU. In 4 of the 13 samples we detected HHV‐7 reads in the respiratory

swabs which may have contributed to the observed disease patterns. In addition, we found Torque

Teno virus and bacteriophage reads. Identified Streptococcus pneumoniae phage reads suggested a

previously not defined infection. Metagenomic approaches can identify microbial pathogens in a single

analysis. Our study confirmed low‐level infections with known respiratory viruses and additionally

identified cases of HHV‐7 infection. No other viruses were found in the remaining symptomatic lung

transplant patients leaving the exact aetiology of infection still unclear. This study highlights the

potential of metagenomic sequencing in complex diagnostic situations such as in

immunocompromised hosts.


14

Regulation of MHC class I endocytosis by the macroautophagy machinery

Loi M.1, Gannagé M.1,2, Lippmann A.1, Becker A.3, Dengjel J.3, Münz C.1

1Viral Immunobiology, Institute of Experimental Immunology, University of Zurich, Switzerland; 2Department of Pathology and Immunology‐School of Medicine, University of Geneva, Switzerland; 3Freiburg Institute for Advanced Studies (FRIAS), School of Life Science (LifeNet), University of Freiburg, Freiburg, Germany.

[email protected]

Macroautophagy is a catabolic process that maintains cellular homeostasis by targeting cellular

organelles and cytoplasmic constituents for lysosomal degradation. Additionally, it is involved in

various aspects of immunity including the clearance of pathogens, survival of immune cells and,

importantly, in pathways of antigen presentation. Recently, it has been described to play a role in the

trafficking of plasma membrane proteins.

Here, we show that immortalized macrophages and dendritic cells (DCs) derived from mice

conditionally depleted for an essential macroautophagy protein in their DC compartment (CD11c‐cre

x ATG5fl/fl mice) express increased MHC class I cell surface levels compared to their autophagy

sufficient counterparts. While MHC class I expression and antigen processing seem unaffected, MHC

class I molecules are stabilized on the cell surface of macroautophagy deficient cells due to decreased

internalization and degradation. Moreover, in ATG5+/+DCs treated with chloroquine the total amount

of MHC class I protein is stabilized, significantly reducing the difference in cell surface MHC class I

expression between ATG5‐/‐ and ATG5+/+ cells. This strongly suggests a degradative role of

macroautophagy proteins for MHC class I molecules after internalization.

More interestingly, macroautophagy‐driven stabilization of MHC class I in antigen presenting cells

enhances CD8+ T cell priming during influenza A virus infection in ATG5CD11c mice, resulting in

decreased pathology.

We are currently characterizing the mechanisms that lead to MHC class I stabilization in the absence

of macroautophagy. The understanding of this process may provide important implications for

therapeutic approaches aimed at improving the adaptive immune response against viral pathogens.


15

Fungal determinants govern the host response to c.albicans in the oral mucosa

Franziska Schönherr1,2, K. Trautwein‐Weidner1,4, F. Kirchner1,4, A. Gladiator1,4, C. Fragoso‐Corti2, O. Petrini3 and S. LeibundGut‐Landmann1

1Institute of Microbiology at ETH Zürich and Institute of Virology at University of Zürich, Switzerland; 2Laboratorio di microbiologia applicata, SUPSI, Bellinzona, Switzerland; 3POLE Pharma consulting, Breganzona, Switzerland; 4equal contribution

[email protected]

The opportunistic fungal pathogen Candida albicans is a member of the normal human microbiota, but

it can cause severe infections in immunocompromised individuals. It is generally believed that the host

immune status determines the outcome of the interaction between the fungus and the host, resulting

in either health or disease. IL‐17 mediated immunity has emerged as a critical mechanism of the host

to regulate the antimicrobial response, thereby limiting fungal overgrowth at the epithelial barriers.

Complementarily, neutrophils contribute to host defense by preventing systemic dissemination of the

fungus.

In a mouse model of oropharyngeal candidiasis the IL‐17 and neutrophil responses are strongly

activated during infection with C. albicans strains SC5314 and both mechanisms account for the rapid

control of the fungus. We explored how the capacity of the fungus to induce these characteristic

responses can affect the infection dynamics. For this, we compared the host response to different

clinical isolates of C. albicans and found that weak IL‐17 and neutrophil responses resulted in persistent

colonization of the oral mucosa by C. albicans and in delayed fungal clearance. However, IL‐17 and

neutrophils remained essential for preventing fungal outgrowth. These findings thus emphasize how

important fungal determinants are to define the host‐pathogen interaction in vivo, which regulates

host colonization versus infection.


16

Host GTPase machinery implicated in the formation of Legionella‐containing vacuoles

Bernhard Steiner1, Christine Hoffmann2, and Hubert Hilbi1,2

1Institute of Medical Microbiology, University of Zurich, Zürich, Switzerland; 2Max‐von‐Pettenkofer‐Institute, Ludwig‐Maximilians‐Universität Munich, Germany.

[email protected]

Undermining host vesicle trafficking machinery is pivotal for survival and pathogenesis of many

intracellular pathogens. One model organism for studying host cell process modulation during

infection is the Gram‐negative accidental human pathogen Legionella pneumophila, which can cause

a severe pneumonia termed Legionnaires` disease. L. pneumophila injects approximately 300

“effector” proteins into host cells through its Icm/Dot type IV secretion system (T4SS), in order to

guarantee intracellular growth in a distinct pathogen compartment termed the “Legionella‐containing

vacuole” (LCV).

Our recent proteomics studies of purified LCVs from infected Dictyostelium discoideum amoebae or

murine RAW 264.7 macrophages identified 13 small GTPases of the Rab family, implicated in the

secretory or endosomal vesicle trafficking pathways. Using fluorescence microscopy, 6 novel Rab

proteins were confirmed to localize on LCVs harboring wild‐type but not ΔicmT mutant L. pneumophila.

Individual depletion of 20 GTPases by RNA interference indicated that endocytic GTPases (Rab5a,

Rab14 and Rab21) restrict intracellular growth of L. pneumophila, whereas secretory GTPases (Rab8a,

Rab10 and Rab32) implicated in Golgi‐endosome trafficking promote bacterial replication. The down‐

stream effectors and functional roles of these GTPases during L. pneumophila infection and LCV

formation are only incompletely understood. The LCV proteomics analysis also suggested that large

GTPases implicated in vesicle fusion and fission are candidate LCV components. Current experiments

aim at the validation of the proteome data and an investigation of the functional roles of these host

factors for L. pneumophila phagocyte infection and LCV formation. Thus, the phagocyte proteomes of

purified LCVs are a valuable resource for further hypothesis‐driven investigations of the complex

process of pathogen vacuole formation.


17

DARPins as Alternative to HIV‐1 broadly Neutralizing Antibodies

Emanuel Stiegeler1, Nikolas Friedrich1, Mustafa Can Eroglu1, Thomas Reinberg2, Mylène Morin3, Yufan Wu2, Jonas V. Schäfer2, John Robinson3, Andreas Plückthun2, Alexandra Trkola1

1Institute of Medical Virology, University of Zurich, Zurich, Switzerland; 2Institute of Biochemistry, University of Zurich, Zurich, Switzerland; 3Institute of Organic Chemistry, University of Zurich, Zurich, Switzerland

[email protected]

Designed Ankyrin Repeat Proteins (DARPins) are small synthetic binding proteins characterized by

exceptional biophysical properties, high target affinities and specificities as well as low production

costs. We previously utilized the DARPin technology to derive HIV‐1 gp120‐V3‐loop reactive clones

with clade B restricted neutralizing activity and a novel, structure‐dependent recognition mechanism

which overcomes HIV envelope shielding. Expanding on our previous finding we here describe the

generation of broadly neutralizing DARPins targeting the V3 loop and membrane proximal external

region (MPER) within HIV‐1 envelope subunit gp41. V3 specific DARPins that possess neutralization

breadths comparable to V3 specific broadly neutralizing antibody (bNAb) PGT121 were isolated. These

clones recognize the V3 loop tip with varying structural preferences. MPER specific DARPins that match

or even exceed the neutralization breadth of bNAbs were generated, though their neutralization

potency still leaves room for improvement. These DARPins bind their target with affinities in the

nanomolar range in a structure dependent manner and act post CD4 engagement to block viral entry.

Our findings underline the potential of the DARPin technology as tool to develop HIV‐1 entry inhibitors

and to gather structural insights on epitope targets which can potentially be used for vaccine design.


18

Poster abstracts

Role of retrograde transport and RidL for intracellular replication of Legionella

Kevin Bärlocher, Ivo Finsel & Hubert Hilbi

University of Zürich, Institute of Medical Microbiology, Gloriastrasse 30/32, CH‐8006 Zürich, Switzerland

[email protected]


19

Structural and functional analysis of lipoprotein glycosylation in fast‐ and slowgrowing mycobacteria

Katja Becker & Peter Sander

University of Zürich, Institute of Medical Microbiology, Gloriastrasse 30/32, 8006 Zürich

[email protected]

With approximately 1,3 million death per year tuberculosis (TB), caused by the bacterial pathogen

Mycobacterium tuberculosis, remains one of the most deadly infectious diseases worldwide (WHO). It

is estimated that 1/3 of the world`s population is latently infected with the bacterium which possesses

a unique and almost impermeable cell envelope. Lipoproteins are abundant in this mycobacterial cell

envelope which enables M. tuberculosis to persist in its human host. They fulfil crucial roles in cell

homeostasis and virulence. Next to their lipidation, many mycobacterial lipoproteins possess glycan‐

moieties but the structure and functions of this second post‐translational modification are unknown.

LppX, LpqW and RpfB are glycosylated lipoproteins involved in cell wall synthesis, virulence and

resuscitation. They will be used as a model for the about 100 lipoproteins in M. tuberculosis. In this

project, the glycosylation sites and structures of these mycobacterial model proteins will be

determined. The function of specific glycan‐moieties on protein localization, stability and function will

be elucidated by mutating glycosylation sites and by assessing the recombinant bacilli in functional

assays. M. smegmatis, a fast‐growing, saprophytic mycobacterium, will be used to identify

glycosylation sites. For functional assays, however, the mutated proteins will be expressed in M. bovis

BCG, a slow‐growing mycobacterium closely related to M. tuberculosis. Virulence assays will be

performed in human macrophages with the M. bovis BCG strains deficient for lipoprotein glycosylation

at specific sites. Results obtained during this project will contribute to the general understanding of

the regulation of cell wall integrity and host‐pathogen interaction.


20

In vivo imaging of Salmonella invasion by electron cryotomography

Desiree Böck

ETH, D‐Biol, Institute of Molecular Biology and Biophysics, Otto‐Stern‐Weg 5, HPK D9, 8093 Zürich

[email protected]

Salmonella is an intracellular pathogen that causes significant morbidity and mortality in both humans

and animals. Salmonellosis is one of the most common food‐borne diseases worldwide, however, its

underlying molecular mechanisms are still not completely understood.

The aim of this project is to provide a better understanding of Salmonella invasion into eukaryotic host

cells – a critical step during eukaryote infection – using electron cryotomography (ECT). ECT is cutting‐

edge electron microscopy technique that allows imaging of cells in a near‐native state. Besides, it has

the potential to bridge and integrate observations from structural (atomic scale) and infection biology

(micron scale). Our plan is first to establish a workflow to allow for imaging of eukaryotic cells. This

approach will then be used to investigate the individual stages of Salmonella invasion.

Salmonella wild‐type and mutants will be imaged to elucidate the number of T3SS per cell and their

polarity towards the host cell. A specific mutant that stalls invasion at the point of host attachment

will be used to study T3SS‐contact sites on the host cell and the translocon structure. In particular, a

desired outcome is to image invasion at the point of bacterial attachment and entry to visualize actin

cytoskeletal rearrangements in the host cell.

This interdisciplinary approach has the potential to produce groundbreaking insights into Salmonella

pathogenesis. Furthermore, it could also prove useful for the investigation of bacterium‐eukaryote

interactions involving other pathogens or symbionts in the future.


21

Expression and characterization of the Plasmodium falciparum & Entamoeba histolytica

Oligosaccharyltransferase

Nathalie Cornillie & Markus Aebi

Institut f. Mikrobiologie, HCI F 412 Vladimir‐Prelog‐Weg 1‐5/10 8093 Zürich

[email protected]

N‐linked glycosylation is one of the most common covalent protein modifications in eukaryotes and

occurs in all three domains of life. This highly conserved process takes place in the endoplasmic

reticulum (ER), where the central step is catalyzed by a large transmembrane protein complex, called

the oligosaccharyltransferase (OST). In eukaryotes, the OST mediates the transfer of a pre‐assembled

lipid‐linked oligosaccharide (LLO) to asparagine residues on nascent polypeptides within the consensus

sequence asparagine‐X‐serine/threonine (N‐X‐S/T), where X can be any amino acid except proline. This

research project focuses on the OST complexes of Plasmodium falciparum and Entamoeba histolytica,

which are predicted to consist of four subunits (Stt3, Wbp1, Ost2 and Ost1). The objective of the

project is to express the OST complexes using the baculovirus/insect cell system, and to purify the

complexes for functional and structural characterization. E. histolytica and P. falciparum differ with

respect to the N‐glycans that are added on proteins in the ER. P. falciparum is missing most of the

glycosyltransferases involved in assembly of the lipid‐linked precursor. Therefore, the OST transfers an

oligosaccharide consisting of only one or two GlcNac residues. In E. histolytica the N‐glycans are

unprocessed Man5GlcNac2 structures lacking a terminal glucose residue, which is a critical substrate

recognition determinant for the OST. The enzymatic activity of the OST complexes will be characterized

by an in vitro N‐glycosylation assay using synthetic fluorescently labeled acceptor peptides and LLOs.

Finally, the OST complexes will be used in structural studies with cryo‐electron microscopy.


22

The evolution of N332 glycan site directed broadly neutralizing antibodies in HIV infection

Ebner Hanna1, Rusert Peter1, Schanz Merle1, Huber Michael1, Kuster Herbert2, Weber Jacqueline1, Uhr Therese1, Zagordi Osvaldo1, Braun Dominique2, Günthard Huldrych F.1, 2, Trkola Alexandra1

1University of Zürich, Institute of Medical Virology, Winterthurerstrasse 190, 8057 Zürich; 2University Hospital Zürich, Division of Infectious Diseases and Hospital Epidemiology, Rämistrasse 100, 8091 Zürich

[email protected]

Broadly neutralizing antibodies (bnAbs) are considered as vital components of protective vaccines

against HIV‐1 and potential HIV‐1 therapeutics. In natural infection antibodies with broad neutralizing

activity are elicited only in 10‐20% of patients. Understanding the parameters that steer bnAb

responses is thus needed to guide vaccine design. Here we report on bnAb evolution in a participant

of the Zurich Primary HIV Infection study. The patient, ZPHI46, had a slow progressing subtype B

infection and maintained a viral set point of 16600 RNA copies/ml and a geometric mean CD4 T‐cell

count of 450 cells/μl over a period of 9 years in absence of antiretroviral therapy. Longitudinal

assessment of plasma neutralizing antibody activity against autologous virus isolated from consecutive

time‐points showed a prototypic picture of continuous escape. Interestingly, the patient developed

high heterologous breadth neutralizing 70% (29 of 42) of virus isolates from different genetic subtypes

at plasma titers higher than 1:100. Autologous virus isolates resistant to contemporaneous plasma

lacked the N332 glycosylation site, a key bnAb‐target, raising the possibility that bnAb activity in ZPHI46

is directed to this site. Comparison of neutralization activity against virus strains that either contain or

lack the N332 glycan revealed dependence of ZPHI46 activity on this glycan. To study the ontogeny of

this bnAb we have isolated monoclonal antibodies. One clone, ZPHI46.1, proved to capture the N332

restricted potency of the plasma response. A detailed analysis of breadth, epitope specificity and

evolution of this bnAb by NGS based Ig repertoire tracking is underway.


23

Asymmetric cell division and barrier function in T cell fate determination Hyuliya Ismail Emurla, Annette Oxenius HCI, Wolfgang‐Pauli‐Strasse 10/Vladimir‐Prelog‐Weg 4, CH‐8093 Zurich, Switzerland [email protected] T lymphocyte is the class of immune cells conduct adaptive immune responses. T cells express antigen

specific surface receptors called TCRs. Antigen presentation driven clonal expansion that drives cell

differentiation and cell proliferation, leads development of population of T cells exerting different

transcription profiles in addition to different cellular functions. Asymmetric cell division is a proposed

mechanism to explain differential fate acquisition of T cells as early as first mitotic division followed by

activation of naïve T cells. Asymmetry introduced by formation of stable immune synapse that includes

immune synapse components in addition to polarity associated factors. T‐bet, a master regulator of T

cell differentiation is showed being unequally partitioned by unequal proteasome localization in APC

distal, and APC proximal daughter cells trough asymmetric cell division. The mechanism, called

“Diffusion Barrier”, showed to be responsible for ER compartmentalization between mother and bud

in yeast through asymmetric cell division. In this study, whether T cells form diffusion barrier through

asymmetric cell division, whether diffusion barrier is involved in unequal partitioning of cell fate

determination factors, and whether it is possible to modulate the strength of the barrier or diffusion

barrier function in general respect to the purpose of manipulating determination of cell fate in naïve T

cells, as well as differentiated subtypes of T cells are the focus of extensive scientific examination.


24

Purification and characterization of the octameric oligosaccharyltransferase from Saccharomyces cerevisiae

Jillianne Eyring & Markus Aebi

Institut f. Mikrobiologie, Vladimir‐Prelog‐Weg 1‐5/10, 8093 Zürich

[email protected]

Asparagine‐linked glycosylation (N‐glycosylation) of proteins is one of the most common covalent post‐

translational protein modifications in eukaryotes and is highly conserved throughout all three domains

of life. The central step of N‐glycosylation is catalyzed by the oligosaccharyltransferase (OST) enzyme

which transfers a pre‐assembled lipid‐linked oligosaccharide (LLO) onto an asparagine residue on

proteins, most commonly within the conserved consensus sequon Asn‐X‐Ser/Thr, where X can be any

amino acid except for proline. In animals, plants and fungi, OST is a heterooligomeric complex

consisting of up to eight non‐identical subunits, all of which are integral membrane proteins. The OST

complex from the budding yeast Saccharomyces cerevisiae is composed of the eight subunits Ost1,

Ost2, Ost4, Ost5, Stt3, Swp1, Wbp1 and, either Ost3 or Ost6. Thereby, two isoforms of the yeast OST

complex exist which are defined by the presence of either Ost3 or Ost6. The aim of this project is to

characterize the yeast OST complex biochemically and structurally to further understand N‐

glycosylation by an oligomeric OST. To purify the OST complex, one of the eight OST subunits will be

tagged in strains expressing only one isoform of OST. Glycosylation efficiency and complex stability will

be analyzed in vivo to screen for the most functional tagged OST complex to be purified. OST enzyme

activity and substrate selectivity will be investigated through in vitro glycosylation assays using

chemically synthesized LLOs and fluorescently labeled peptides as substrates. Finally, structural studies

will be done by cryo‐electron microscopy.


25

Glycoengineering in insect cells to produce carbohydrate‐based vaccines against Haemonchus contortus

Susanna Fleurkens1, 2, Chia‐Wei Lin1, Alex Butschi2, Bruno Oesch2, Markus Aebi1

1Institute of Microbiology of ETH Zürich, Switzerland; 2Malcisbo AG, Schlieren, Switzerland

[email protected]

The blood sucking stomach worm of sheep Haemonchus contortus causes major economic damage

reducing meat and wool quality. The parasite is currently controlled by chemical treatments yet the

advent of resistant strains necessitates new strategies such as an effective vaccine. It is well known

that glycoprotein‐enriched extracts of adult H. contortus are able to confer immunity thereby reducing

the worm burden. However, attempts to make a recombinant vaccine have failed up to now which

might be due to the lack of glycosylation.

We therefore manipulate the insect N‐glycosylation pathway by overexpressing additional

glycosyltransferases enabling the production of various antigens of H. contortus carrying parasite

specific glycan structures. Here we report the results of the co‐expression of the major antigen H11

and the nematode‐specific galactosyltransferase using High five insect cells. A novel mass

spectrometry analysis tool for identification of site‐specific N‐glycosylation reveals comparable

glycosylation patterns for H11 expressed in “glyco‐modified” insect cells and native H11 extracted from

adult worms. This indicates that we have been able to produce a potential immunogen for a H.

contortus vaccine in large quantities in order to test the hypothesis of the importance of glycosylation.


26

Characterization of Mycobacterium smegmatis PafBC

Begonia Fudrini, Sibylle Burger, Frank Imkamp

University of Zurich, Institute of Medical Microbiology, Zurich, Switzerland Gloriastrasse 30/32 CH‐8006 Zürich

[email protected]

PafBC is a heterodimeric protein which is encoded by genes that are organized in an operon with the

Pup‐ligase PafA, which is part of the Pup‐proteasome system (PPS) present in mycobacteria and other

actinobacterial species. The PPS is crucial for Mtb resistance towards reactive nitrogen intermediates

(RNI). PafBC however seems to play only a minor role in RNI resistance. PafBC is a heterodimeric

protein and our bioinformatic analysis revealed a helix‐turn‐helix DNA binding motif at the protein’s

C‐termini, implying a function for PafBC as a transcriptional regulator. The current project aims is to

understand the physiological role of PafBC. We therefore generated a pafBC deletion mutant in the

model organism Mycobacterium smegmatis. We will perform a comprehensive analysis of the mutant

under various growth conditions in order to gain initial insights regarding the function of PafBC.

Furthermore, we will conduct iTRAQ analyses in order to detect PafBC dependent proteome changes,

which will provide important information about PafBC regulated pathways.


27

Correlative Multi‐Modal Imaging for 3D Spheroid Analyses Fanny Georgi, Vardan Andriasyan, Artur Yakimovich, Urs Greber University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse 190, 8057 Zürich [email protected] Spheroids are self‐assembled 3‐dimensional (3D) spherical cell aggregates. They are in vitro micro‐scale

tissue mimetics, and can be used for exploring physiological features of healthy or cancerous tissue.

They provide biological complexity and have been shown to be superior assay platforms compared to

monolayer cell culture systems. Moreover, novel techniques allow cultivation of multiple cell types as

spheroids of defined sizes suitable for high‐throughput screening (HTS) assays. Spheroid‐based assays

are used for drug discovery and can be exploited for therapeutic strategies employing oncolytic viruses.

The cellular processes underlying spheroid formation and growth are poorly understood. In addition,

current spheroid‐based HTS assays are largely based on cell population measurements, and therefore

ignore spheroid system complexity. Here, we aim to provide spatial information on molecular markers

and other biological features concealed in spheroid HTS assays to improve accuracy and reliability of

spheroid‐based screens. We present a correlative multi‐modal imaging approach enabling hit

refinements and characterization of molecular properties of a spheroid. We propose live high‐

throughput transmission light imaging as a HTS assay for monitoring spheroid assembly, maturation,

viability and growth. This assay is complemented by a quantification framework characterizing

spheroids using morphological and textural features. Assay endpoints are further characterized in

chemically fixed spheroids employing lightsheet microscopy to 3D‐render spheroids tomograms. We

show that the combination of these modalities is feasible in a correlative funnel‐like manner, which

may allow precise HTS hit refinement. We further show applicability of this approach to study infection

of carcinotypic spheroids by an oncolytic human adenovirus.


28

The influence of Alzheimer’s Disease‐like amyloid pathology on the immune surveillance in the brain

Christoph Gericke, Claudia Späni, Nora Schweizer, Mario Merlini, Tobias Suter, Luka Kulic, Roger M. Nitsch, Maria Teresa Ferretti

Division of Psychiatry Research and Psychogeriatric Medicine, University of Zurich

[email protected]

Objectives. Chronic brain inflammation is a hallmark of Alzheimer’s disease (AD); however, no

protective autoimmunity against amyloid beta peptide (Abeta) is observed so far. Here, we test the

hypothesis that Abeta alters the brain immune surveillance.

Methods. Antigen presenting cell (APC) and T cell numbers and phenotype were studied using ex vivo

analysis via flow cytometry and confocal microscopy. Results from aged transgenic (tg) mouse models

over‐expressing amyloid precursor protein were compared with non‐tg, age‐matched littermates.

Results. In SweArc‐tg mice we have observed reduced major histocompatibility class II (MHC‐II) surface

expression on APCs. Further analysis has revealed that most MHC‐II accumulated intracellularly,

suggesting an immature APC phenotype. Furthermore, we found a significant increase in brain CD8+ T

cell numbers with low effector cytokine production. We could confirm the altered MHC‐II expression

in brain APCs from a second model, the APP‐PS1deltaE9. T cells here comprised less pro‐inflammatory

cells secreting cytokines such as IFNγ or TNFα. No differences were observed neither in APC from

cerebellum nor splenic T cells in transgenic and control animals, indicating a cerebrum and amyloid‐

specific effect.

Conclusions. Our preliminary results suggest that amyloid accumulation in the brain is accompanied

by significant alterations in both MHC‐II expression and T cell cytokine production. Further studies

using both basic in vitro and in vivo approaches are underway to explore the possibility that brain

immune surveillance is defective in the context of AD‐like amyloid pathology.


29

Signaling in the General Stress Response of Sphingomonas melonis Fr1

Lisa Gottschlich, Sebastian Dintner, Julia Vorholt

Institute of Microbiology, ETH Zürich, Vladimir‐Prelog‐Weg 1‐5/10, 8093 Zürich, Switzerland

lisa‐[email protected]

The phyllosphere represents a harsh environment characterized by constantly changing conditions.

Colonizing bacteria are able to counteract different stress stimuli with a specific as well as a so called

general stress response (GSR). The latter leads to cross‐protection against a variety of different

stresses. In Alphaproteobacteria, the PhyR‐NepR‐σecfG cascade is the central mechanism of GSR. A

stress stimulus sensed by different histidine kinases leads to phosphorylation of the anti‐sigma factor

antagonist PhyR. After subsequent conformational changes, PhyR forms a complex with the anti‐sigma

factor NepR. Therefore, the previously bound alternative extracytoplasmic function sigma factor (ECF)

is released and able to bind RNA polymerase, which leads to differential gene expression. This

conserved partner switching mechanism is also called "mimicry effect".

The aim of this project is to achieve further understanding of GSR in Sphingomonas melonis Fr1, a

gram‐negative, epiphytic Alphaproteobacterium. It is of great interest to gain insight into stress

induced changes of the transcriptome. Therefore, next generation sequencing will be used to compare

mRNA levels of knockout mutants with impaired GSR compared to wildtype under different stress

conditions. Furthermore, the question will be addressed, if there is a "general stress memory" meaning

a dependency of GSR on duration or frequency of stress exposure. It is also essential to analyze the

mode of action of the single domain response regulator SdrG, which has been found to be a positive

regulator of GSR, in order to obtain a more complete picture of that complex signal transduction

pathway.


30

Dynamic 13C labeling reveals in vivo half‐life of organic coenzymes in exponentially growing cells

Hartl Johannes, Meyer Fabian, Kiefer Patrick & Vorholt Julia

ETH Zürich, Department of Microbiology, Vladimir‐Prelog‐Weg 1‐10/5, 8093 Zürich, Switzerland

[email protected]

A typical cell mainly consists of proteins, nucleic acids, lipids, and to a large proportion, small molecules

(i.e. metabolites). If not acquired from the environment, these constituents are constantly synthesized

and turned over in order to suit both the anabolic and catabolic demand of growing cells.

The turnover of macromolecules has recently received considerable attention, and it has been shown

that proteins can be stable for several generations in microorganisms. In contrast, metabolite pools

typically exchange within seconds. Major factors limiting the half‐life of metabolites are their rapid

consumption, degradation pathways, chemical reactivity and instability, or loss into extracellular

space. Instead, few of these aspects hold for organic coenzymes. They are often end‐point reactions

of biochemical pathways; act as catalysts, and are rarely consumed. Indeed, it has been shown that

some organic coenzymes can be surprisingly long‐lived.

Here, we employed high resolution mass spectrometry and 13C dynamic labeling experiments to

investigate the in vivo half‐life of organic coenzymes in the model organisms E. coli, B. subtilis and S.

cerevisiae. We were able to detect and quantify the intracellular fractional labeling of eight organic

coenzymes, and find that most have a half‐life that is very close to the doubling time of the respective

microorganism. Even after more than three generations, we were still able to detect coenzymes in

which no carbon exchange had occurred. Taken together, our data suggests that despite their

reactivity, most coenzymes are in vivo stable enough to allow carbon exchange close to the minimal

necessary rate, de‐novo synthesis mainly counters dilution by cell proliferation, and

salvage/degradation only plays a minor role.


31

Hunt for somatic mutations in Linear Localized Scleroderma

Rebecca Higgins1,2, Martin Theiler1,2, Alexandra Smith1,2, Regula Wälchli1,2, Lisa Weibel1,2 and Alexander A. Navarini1

1Department of Dermatology, University Hospital Zurich; 2Department of Dermatology, University Children’s Hospital Zurich.

[email protected]

Linear localized scleroderma (LLS) is a rare connective tissue disorder characterized by chronic

inflammation and accumulation of collagen. This then results in both hardening and thickening of the

skin leading to the affected areas to cave in from atrophy. It can affect patients in areas throughout

the body including arms, legs and in more rare cases the face. This leads to terrible disfigurement that

cannot be concealed. The disease is limited in treatment options and most often these treatments are

unsatisfactory. The incidence of localized scleroderma in adults and children is 2.7/100’000 population

per year classifying it as a rare disease. LLS is characterized by sharply delimited and linear plaques that

are oriented along embryonal cellular migration routes. This suggests that LLS could be caused by a de

novo somatic mutation(s) resulting in a mosaic state as has been described previously in other skin

conditions. The aim of this project is to use whole exome sequencing to find the causative mutation(s).


32

From protein function to supermolecular structure: Listeria phage A511 baseplate structure and identification of receptor binding proteins

Mario Hupfeld1, Ricardo Guerrero‐Ferreira2 , Martin Loessner1, Takashi Ishikawa3, Petr Leiman2 and Jochen Klumpp1

1Institute of Food, Nutrition and Health Schmelzbergstrasse 7, ETH Zurich, 8092 Zurich, Switzerland; 2Institut de Physique des Systèmes Biologiques, EPF Lausanne, LBBS, Lausanne, Switzerland ; 3Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen PSI, Switzerland

[email protected]

Our understanding of the infection of Gram‐positive bacteria by phages is still incomplete.

Bacteriophage A511 is a contractile‐tailed, dsDNA, Myoviridae phage with a wide host range within the

genus Listeria. It has been shown to be a good tool in pathogen detection especially in food products.

The first steps in the infection cycle are initiated by the attachment to the surface of the bacterium.

This is believed to be a two‐step process, mediated by two different receptor binding proteins (RBPs).

We could identify one RBP, namely Gp108, supposedly a short tail fibre protein of the phage. Here we

show the results of GFP binding assays carried out for the other protein candidates that come into

contact with the bacterial cell surface upon infection. These include Gp98 and Gp99 which form the

central hub‐like structure of the baseplate. Gp104 is similar to a putative tail fiber, although a function

is not known. Gp106 represents a VrlC like protein, which has been found in a number of phages

infecting both Gram‐positive and Gram‐negative bacteria and is considered a potential receptor

binding protein of phage A511. Future experiments are aimed to further characterize these proteins.

We are also developing a structural model of the A511 baseplate (in the contracted and non‐

contracted state) which will be based on Cryo‐electron microscopy images fitted with crystallized

protein structures. The expected results will elucidate the mechanisms involved in phage contraction,

adsorption and subsequent viral DNA injection.


33

Dynamics of the bone‐marrow microenvironment during viral infections

Stephan Isringhausen, Nike Kraeutler, Diana Stoycheva, Ute Suessbier, Markus Manz, Annette Oxenius, Cesar Nombela‐Arrieta

Experimental Hematology, University of Zurich, Zurich

[email protected]

Hematopoiesis, the primary function of the bone‐marrow (BM), is a highly dynamic and tightly

regulated process resulting in the production of millions of blood cells per minute under homeostatic

conditions. Continuous hematopoietic cell production is sustained by a rare population of self‐

renewing, multipotent hematopoietic stem and progenitor cells (HSPCs), which reside in specialized

nurturing microenvironments within BM cavities. The BM ecosystem is extremely diverse, comprising

all hematopoietic lineages as well as complex stromal cellular networks of mesenchymal, neural and

vascular origin. Beyond providing a structural scaffold for hematopoietic cells, stromal cells are

critically involved in the fine regulation of different stages of hematopoiesis. Yet, the functional

interplay of stromal components with hematopoietic cells during both homeostasis, as well as in

inflammatory conditions is still incompletely understood.

Bacterial and viral infections trigger major stress in the hematopoietic system inducing an adaptative

response in cellular output. Surprisingly, little is known about the potential damage that potent

inflammatory stimuli can inflict on the BM stroma, resulting in long term dysfunctional hematopoiesis.

Here we combine established in vitro and in vivo assays with cutting‐edge 3D imaging technologies

recently established in our lab, to study the effect of acute and chronic viral infections in the

microarchitectural and functional integrity of the BM.

Our results indicate that viral infections result in rapid vasodilation of BM sinusoids, loss of extracellular

matrix networks and regional emergence of adipocytes. Notably, chronic infections with LCMV clone‐

13 and docile strains induce a profound and sustained reduction in the number of HSCs, which

correlate with a downsizing of the population of mesenchymal stromal progenitor cells and a decrease

in their capacity to produce HSPC‐sustaining factors such as CXCL12. We are currently investigating the

precise molecular and cellular mechanisms driving viral‐induced BM tissue damage.


34

Conformational protection of antibody epitopes on native HIV‐1 envelope glycoprotein trimers

Branislav Ivan1, Carsten Magnus1, Oliver F. Brandenberg1, Roland Regoes2, Peter Rusert1, Alexandra Trkola1.

1Institute of Medical Virology, University of Zurich, Switzerland; 2Institute of Integrative Biology, ETH Zurich, Switzerland

[email protected]

Antibodies binding non‐native forms of the HIV‐1 envelope glycoprotein (Env) appear early in infection

and are also routinely elicited in animal and human vaccination trials. However, such antibodies fail to

bind the functional Env trimer and to neutralize most HIV‐1 isolates. A major driver of antibody

resistance of the Env trimer is the V1V2 domain of gp120 which shields neutralization sensitive

epitopes of gp120, in particular the V3 loop. Two modes of V3 loop shielding by the V1V2 loop have

been proposed: I) self‐protection where both V3 and V1V2 are located on the same protomer within

the Env trimer and II) neighboring‐protection where the V1V2 loop from one protomer shields the V3

loop on the neighboring protomer. Thus far structural and functional studies of the HIV‐1 envelope

yielded inconclusive or even seemingly contradicting results concerning the mode of V3 protection by

the V1V2 loop raising the possibility that both protection modes may occur depending on the envelope

and epitope studied.

To probe this, we tested the binding of a range of V3 directed antibodies and inhibitors to envelope

proteins from divergent HIV strains in the context of mixed HIV Env trimers containing antibody binding

sensitive or resistant V3 loops in presence or absence of the V1V2 loop. Employing a mathematical

model we previously described that predicts antibody binding to mixed trimers allows us to assess the

stability of mixed trimers and dissect the mode of V1V2 loop protection.


35

Response to nitrogen limitation in Burkholderia phymatum and competition between β‐rhizobia for legumes infection

Martina Lardi1, Gabriela Purtschert1, Maria Sanchez‐Contreras1, Alessandro Pedrioli1, Yilei Liu1, Christian Ahrens2, Leo Eberl1 and Gabriella Pessi1

1Institute of Plant Biology, Department of Microbiology, UZH Zurich, Switzerland; 2Institute for Plant Production Sciences, Agroscope, Wädenswil, Switzerland

[email protected]

Rhizobium‐legume symbiosis is of major ecological and economic importance and accounts for two‐

thirds of the nitrogen fixed globally. Until recently, all known examples of symbiotic relationships

between legumes and prokaryotes were confined to the phylogenetically diverse α‐rhizobia. This

changed with the discovery of certain β‐proteobacteria of the genera Cupriavidus and Burkholderia,

which are also capable of establishing nitrogen‐fixing symbiosis with legumes. At present, very little is

known about molecular determinants underlying the successful establishment of the symbiosis

between legumes and β‐rhizobia.

In rhizosphere, rhizobia are subject to changing environmental conditions, including nutritional

stresses. It has been shown that under nitrogen limited conditions β‐rhizobial strains such as

Burkholderia phymatum STM815 are the most competitive symbionts.

In this study, we used RNA‐Sequencing to investigate the transcriptome of B. phymatum in response

to nitrogen limitation. Among the genes responding significantly to nitrogen limitation, we found

several genes for nitrogen assimilation. In addition we found also genes coding for proteins involved

in polyhydroxybutyrate accumulation as well as exopolysaccharide synthesis. We next investigated the

capacity of seven β‐rhizobial strains to compete for nodulation of different legumes. We found B.

phymatum to be the most competitive strain on Vigna unguiculata, and Burkholderia tuberum was the

most successful strain on Macroptilium atropurpureum. Phaseolus vulgaris was found to be

preferentially nodulated by B. tuberum and B. phymatum. While Mimosa pudica was nodulated by B.

phymatum and Burkholderia mimosarum. The identification of genes important for competiveness

would allow us to better understand natural selection for high nitrogen fixation capacity.


36

Infection‐induced changes in regulatory T cells

Katharina Littringer & Nicole Joller

Institute of Experimental Immunology, University of Zürich, Winterthurerstrasse 190, 8057 Zürich, Switzerland

[email protected]

CD4+ Foxp3+ regulatory T cells comprise a highly heterogeneous population of immune modulatory

cells essential for maintaining immune homeostasis. In the course of infections Tregs differentiate into

phenotypically and functionally diverse subsets, parallel to effector T helper (Th) cell responses.

Activated Tregs mirror their Th‐counterparts in their expression of lineage specific transcription factors

such as Tbet (Th1), IRF4 (Th2) or Stat3 (Th17) as well as specific chemokine receptors. This

specialization seems to enable selective control of ongoing immune responses and Treg recruitment

to the site of inflammation. Nevertheless, how the Treg compartment is altered by infections triggering

distinct types of Th responses is still only poorly understood. Not only the nature of Tregs (natural vs.

induced) and their phenotypic changes but especially how their suppressive capacity is affected during

an infectious challenge will be the focus of our work. We address this question in the setting of an

acute infection with Lymphocytic Choriomeningitis Virus (LCMV) or Candida albicans, which generate

a polarized Th1 or Th17 immune response, respectively. Our experiments indicate that Treg activation

in mice, infected with LCMV peaks at day 10 to 14 p.i. Within this timeframe surface receptors and

markers, known to promote Treg specific function, such as CXCR3, CTLA‐4, TIGIT, PD‐1 are highly

upregulated when compared to naïve mice as assessed by flow cytometry. How functionality of this

activated population is changed in those settings will be investigated in a next step using in vitro

suppression assays.


37

Investigation of factors influencing pC3 stability within the Burkholderia cepacia complex Olga Mannweiler, Dr. K. Agnoli‐Antkowiak and Prof. L. Eberl Department of Microbiology, University of Zürich, Zollikerstr. 107, Switzerland [email protected] The Burkholderia cepacia complex (Bcc) is a group of 18 closely related but metabolically versatile

bacterial species, which are able to occupy a variety of environmental niches. Strains of the Bcc are

known as important, life‐threatening opportunistic pathogens for immunosuppressed patients and

individuals suffering from cystic fibrosis. Bcc members harbour three large replicons of which 2 are

chromosomes carrying essential genes and a nonessential megaplasmid, pC3, associated with

virulence and both antifungal and proteolytic activities. It has been shown that it is possible to delete

pC3 from most Bcc members using a plasmid incompatibility approach. The mobilization of pC3 and

therefore its transfer by conjugation was achieved, but this was only possible between certain Bcc

members.

The objective of this project is to investigate the factors that may cause the transfer difficulties

between the Bcc strains by phenotypical, biochemical and functional genomics analyses. Our current

hypothesis is that this might be due to the effect of restriction‐modification (RM) systems. Rebase ®,

the restriction enzyme database, shows that there is a variety in the RM systems for all sequenced

members of the Bcc. Sequencing of the Bcc strains by using the PacBio SMRT technology and

determining the methylome will allow us to compare their methylation patterns, giving us an indication

of how to improve pC3 transfer. Another approach refers to the presence of potential contact‐

dependent growth inhibition systems and whether they affect the pC3 transfer. To test this possibility,

competition assays will be performed


38

Glyco‐conjugate vaccine against Dirofilaria immitis

Francesca Martini1, 2, Chia‐wei Lin2, Alex Butschi1, Bruno Oesch1, Markus Aebi2

1Malcisbo AG, Schlieren, Switzerland; 2Institute of Microbiology of ETH Zürich, Switzerland

[email protected]

Dirofilaria immitis is a parasitic nematode of canidae and the primary cause of cardiopulmonary

dirofilariosis in dogs, cats and ferrets, presenting also zoonotic potential. This parasite is currently

controlled by chemical treatments which prevent the infection. The preventive treatment presents

several disadvantages, the drugs are highly toxic for the dogs and very expensive. Furthermore, in

recent years several cases of anthelmintic resistances have been reported, which indicates the need

for new strategies to control this infection. The goal of this project is to develop the basis for a novel

vaccine against the blood‐sucking nematode Dirofilaria immitis in dogs. To date, no potential vaccine

targets have been found in D. immitis. However, several promising vaccine candidates of other blood

sucking nematodes have been published, for example of Ancylostoma caninum, the dog’s hookworm.

Searching the D. immitis genome for homologous A. caninum antigens might therefore be a valuable

strategy for vaccine target identification. Moreover, in recent years it has become evident that the

protein backbone of an antigen is not the only factor eliciting a protective immune response, also

secondary structure and post‐translational modifications, like glycosylation, are to be taken into

account. Thereby our strategy comprises the use of Mass Spectrometry analysis and lectin blotting to

gain information about the glycome of D. immitis at various developmental stages that will be used to

generate a glyco‐conjugate vaccine.


39

N‐linked glycosylation in the Endoplasmatic Reticulum and Golgi

Corina Mathew

Institute of Microbiology, ETH Zurich, HCI, Wolfgang‐Pauli‐Strasse 10/Vladimir‐Prelog‐Weg 4 CH‐8093 Zurich, Switzerland

[email protected]

The N‐linked glycosylation of proteins is one of the most prevalent post‐translational modifications

and ubiquitous among all kingdoms of life. Attached to a protein, N‐glycans contribute to a variety of

biological functions: They are for example crucial for protein folding, contribute to the quality control

that takes place in the endoplasmatic reticulum (ER) and alter the behavior of a protein regarding

protein stability, solubility, immune response and cellular function. These properties make the study

of the N‐glycosylation process of proteins an important task, because not only academics but also the

production of pharmacological relevant glycoproteins could benefit from the generated knowledge.

The complex biosynthesis of glycoproteins includes assembly of an oligosaccharide recursor, transfer

of the precursor onto nascent polypeptide chains, and modification of the glycan in the ER and the

Golgi. The great diversity of glycans observed on secreted proteins is species‐, tissue‐ and cell‐specific

and produced in the Golgi. Here a variety of glycosyltransferases and glycosidases process the initial

sugars. Our group previously showed that the protein structure and its interaction with the covalently

attached glycans are the major determinants of N‐glycan processing. To study the importance of

glycan‐protein interactions we will employ a number of different in vivo and in vitro experiments along

with in slico techniques to further test and develop these findings. Amino acid residues that showed

prolonged interaction with the glycans of the model protein in molecular dynamic studies will be

mutated to test whether these mutations alter N‐glycan profiles on the respective site. If time allows,

we will start to reconstitute in vitro selected glycosylation steps.


40

Identification of a Novel Genetic Cause of Hypereosinophilia

Andrea Mauracher, Jana Pachlopnik, Stefano Vavassori, Lennart Opiz

UZH, Kinderspital Zürich, Pädiatrische Immunologie, August‐Forel Strasse 7, 8008 Zürich

[email protected]

Eosinophils play an important role in the pathophysiology of allergic, parasitic and malignant diseases

and normally only 1‐3% of all peripheral leukocytes are eosinophils. Eosinophils can infiltrate tissues

and cause extensive tissue damage. They can also secrete cytokines that can activate the adaptive

immunity and can act as antigen presenting cells. The proliferation and differentiation of eosinophils,

their survival in the blood and tissue as well as their localization is stimulated by cytokines and growth

factors including IL‐3, granulocyte macrophage colony‐stimulating factor (GM‐CSF), and IL‐5. Activated

T‐cells are believed to be the main producers of these cytokines whereby IL‐5 is the cytokine most

specific for eosinophils. In contrast, IL‐3 and GM‐CSF exhibit more pleiotropic functions.

Understanding the factors that regulate eosinophils is pivotal in developing new therapeutic

approaches. There still are many mysteries in patients presenting Hypereosinophilia and they offer a

good starting point to unravel the functionality of eosinophils further.

We are analysing two patients who both have a very unique phenotype that we believe could provide

further insights into the aetiology of hypereosinophilia. Our hypothesis is that the hypereosinophilias

seen in our patients are due to a novel genetic mutation. Our aim is to identify the mutations and

establish the link between the mutations and the hypereosinophilia by the use of whole exome

sequencing as well as various in vitro methods.


41

Molecular Epidemiology of Transmitted HIV‐1 Drug Resistance in Drug‐Naive and Newly Diagnosed Patients in Cameroon

Herbert A. Mbunkah, Stefan Schmutz, Alex Marzel, Nottania K. Campbell, Roger Kouyos, Günthard Huldrych, Karin J Metzner

Institute address: University Hospital Zurich, Division of Infectious Diseases and Hospital Epidemiology, Rämistrasse 100, CH‐8091 Zurich

[email protected]

The use of combinations of antiretroviral drugs has proven remarkably effective in controlling the

progression of HIV‐1 and prolonging survival, but these benefits can be compromised by the

development of drug resistance. There is very limited data on HIV‐1 drug resistance mutations in our

study site (Cameroon). We plan to determine the prevalence, types and transmission patterns of

resistance‐associated mutations in drug‐naive and newly diagnosed HIV‐1 patients in Cameroon. At

least 300 blood samples will be collected within a period of one year from some hospitals running HIV

treatment centres in Cameroon. Dried blood spot samples will be collected and stored for onward

transmission to Switzerland. For genotype resistance testing, the next‐generation sequencing (NGS)

platform Illumina MiSeq will be used to sequence the protease and reverse transcriptase genes of HIV‐

1 in these clinical samples. A NGS assay suitable for all subtypes will be scaled up to develop an

affordable drug resistance assay which might be suitable for drug resistance testing in resource limited

countries. The assessment of transmitted resistance will be done as described by the International

AIDS Society‐USA (IAS‐USA) and by the list of drug resistance mutations for surveillance of transmitted

HIV‐1 drug resistance, recommended by the World Health Organization (WHO). Phylogenetic trees will

be constructed with Clustal W, using reference pol sequences. Routine baseline information on the

frequency and types of drug resistance mutations in Cameroon will help to inform optimal

antiretroviral therapy and enable the government to better monitor the success of the national AIDS

treatment program.


42

Methylotrophy ‐ From understanding to engineering

Fabian Meyer, Jonas Müller, Patrick Kiefer, Julia Vorholt

Institute of Microbiology, ETH Zurich, Vladimir‐Prelog Weg 4, 8093 Zürich, Switzerland

[email protected]

The ability to use methanol as sole carbon source makes methylotrophs interesting candidates for

industrial applications. The potential of natural methylotrophs such as Bacillus methanolicus has so far

been restricted, due to the lack of tools for genetic manipulation. As an alternative approach this study

has the aim to combine the potential of a methanol based metabolism with the genetic accessibility of

Escherichia coli by engineering a methylotrophic E. coli using synthetic biology. Achieving this goal is

combined with a deep understanding of the system biological properties of natural methylotrophs.

Performing metabolome analysis in the natural methylotroph B. methanolicus might help to identify

methylotrophic specific features needed to create an artificial methanol utilizing E. coli. So far we

managed to create an engineered E. coli strain which is able to significantly incorporate methanol into

different core‐metabolites, up to 40% in case of hexose‐6‐phosphate. The incorporation of methanol

was shown by using dynamic 13C labeling experiments starting from 13C methanol.


43

Fluidic Force Microscopy ‐ A Novel Technology for Single‐Cell Manipulation and Micropatterning

Maximilian C.A. Mittelviefhaus1, Eva Potthoff1, Orane Guillaume‐Gentil1, Tomaso Zambelli2, Julia A. Vorholt1

1ETH Zurich, Institute of Microbiology, Vladimir Prelog Weg 4, CH‐8093 Zurich; 2ETH Zurich, Institute for Biomedical Engineering, Gloriastrasse 35, CH‐8092 Zurich [email protected]

There is increasing interest to manipulate and analyze single cells, e.g. to uncover cell‐cell interactions

or to investigate single cell behavior in complex consortia in a time and space dependent manner.

Fluidic force microscopy (FluidFM) is a recently invented technology that addresses the demand for

novel single‐cell research. By combining atomic force microscopy with microchanneled cantilevers and

microfluidics, FluidFM constitutes a unique technique for local fluid delivery, lithography, single‐cell

manipulation, and micropatterning.

The microchannels in the cantilever form a continuous fluidic circuit between an externally connected

pressure controller and an aperture in the tip of the cantilever. Application of overpressure or vacuum

allows controlled manipulation of liquids within the cantilever. Overpressure, for example, can be

utilized for controlled liquid injection after cell penetration into single cells. By creating a vacuum,

micro‐objects, including single bacterial cells, can reversibly be immobilized on the cantilever and be

used for precise measurements of adhesion forces in a serial manner. By exploiting both, vacuum and

overpressure application, the system allows for pick‐and‐place experiments of single cells. By defined

spatial manipulation of individual bacteria, FluidFM can also be used to create arrangements of

microorganisms to investigate cell‐cell interactions in a time and space dependent manner.

Furthermore, the technology can be used as versatile lithography tool through force and pressure

controlled local polymer delivery. Adhesive surface coatings within a nonfouling background, such as

poly‐L‐lysine (PLL) on a PLL‐g‐poly‐ethylene‐glycol (PEG) background, enable the immobilization of

bacteria and the systematic study of cell‐cell interactions at defined distances.


44

An in vivo study of HIV virus distribution and HIV‐Env specific antibodies in order to eliminate

latently infected cells

Christina Müller

University Hospital Zürich, Rämistrasse 100, 8091 Zürich

[email protected]

Combined antiretroviral therapy (cART) is the current state of the art in treating HIV. cART achieves in

most patients a long‐term suppression of HIV and has a remarkable positive impact on morbidity and

mortality. However, HIV rebounds after interruption of therapy. This rebound is due to long‐lived

latent HIV‐infected CD4+ cells and is one of the major hurdles in eliminating HIV infection. In addition,

the viral diversity of HIV resulting in immune and drug escapes as well as the lack of a vaccine are major

barriers towards finding a cure for HIV. Nevertheless, over the past years, new promising potent and

broadly neutralizing antibodies targeting the HIV envelope (HIV‐Env) have been identified and isolated.

These antibodies could provide a tool to eventually eliminate HIV. Therefore it is important to

investigate the specificities of the antibodies regarding to their ability to control HIV infection in vivo.

Furthermore these antibodies can be used to achieve a better understanding of HIV latency in vivo.

To investigate these two aspects in vivo the humanized mice model provides a good tool and therefore

will be used in this context. The goal is to evaluate the most effective antibodies for therapy. Therefore

HIV‐Env specific antibodies targeting different epitopes will be tested in HIV infected humanized mice.

Furthermore it is the aim to use labeled HIV‐Env specific antibodies in order to follow the virus

distribution during progression of disease by in vivo imaging. Finally, we will study combinations of the

most efficient HIV‐specific antibodies in concert with compounds activating latently infected cells for

their efficient elimination.


45

Anti‐TNF promotes type I interferon‐driven psoriasis‐like skin inflammation

Curdin Conrad1, Jeremy Di Domizio1, Alessio Mylonas1, Cyrine Belkhouja1, Olivier Demaria1, Anne‐Karine Lapointe1, Maxime Vernez1, Alexander Navarini2, Lars French2, Michel Gilliet1

1Departement of Dermatology, CHUV Lausanne; 2Departement of Dermatology, University Hospital of Zurich

Email: [email protected]

Paradoxical psoriasis is a well‐known side‐effect of anti‐TNF therapy affecting 2‐5% of treated patients.

As this side effect often necessitates cessation of the anti‐TNF therapy, there is an urgent need to

understand its pathogenesis. Here, we analyzed a series of 25 cases of paradoxical psoriasis induced

by all anti‐TNF agents available. Underlying diseases, clinical presentation and histological patterns

varied considerably among patients. However, we found a striking, uniform, and selective upregulation

of type I interferons (IFN) in skin of paradoxical psoriasis as compared to classical psoriasis. The

overexpression of type I IFN was paralleled by a massive accumulation of plasmacytoid dendritic cells

(pDCs) within the skin. In‐vitro, TNF blockade directly enhanced type I IFN production by pDCs, while

TNF itself inhibited its production suggesting a crossregulation of TNF and pDC‐derived type I IFN. In a

skin injury mouse model, anti‐TNF therapy increased and sustained type I IFN expression and skin

infiltration by pDCs and was sufficient to induce a psoriatic phenotype in a type I IFN‐dependent

manner. Our study demonstrates that anti‐TNF therapy unleashes unabated type I IFN production by

pDCs, thereby inducing a psoriasis‐like skin phenotype. Thus, we unravel the pathomechanism of

paradoxical psoriasis and provide a clinical relevance for the crossregulation of TNF and type I

interferon.


46

Infection‐Induced Alterations of CD4+ T Cell Responses upon Heterologous Challenge

Nikolas Rakebrandt & Nicole Joller

University of Zürich, Institute of Experimental Immunology, Winterthurer Strasse 190

[email protected]

CD4+ Foxp3+ regulatory T cells (Tregs) are essential in maintaining self‐tolerance. Recent evidence

suggests that they also play a crucial role in modulating the nature of the immune response. Depending

on the challenge, CD4+ effector T cells differentiate into specialized T helper (Th) lineages such as Th1,

Th2 and Th17, which are defined by production of distinct sets of cytokines. In order to cope with the

diversity of CD4+ T cell responses, Tregs show a similar level of heterogeneity and thereby the capacity

to regulate the entire range of effector responses. Emerging evidence suggests the formation of tissue

resident memory Tregs following immune responses. However, whether memory‐dependent changes

in the composition of the Treg compartment affect the nature of secondary challenges is still unclear.

We aim to analyze if a preceding acute infection affects the type or magnitude of the immune response

upon heterologous challenge and whether this is dependent on an altered Treg compartment or

formation of regulatory memory. To address this question, mice are primed with acute lymphocytic

choriomeningitis virus (LCMV), which induces a Th1 response and then challenged, after viral

clearance, with Legionella pneumophila eliciting a mixed Th1/Th17 cell response. We then analyze the

effector CD4+ T cell response and Treg populations using flow cytometry. Overall, these experiments

will allow us to assess if a preceding immune response alters the nature of the immune response upon

heterologous challenge and how this affects susceptibility to pathogenic challenges or autoimmune

diseases.


47

Impact of Nlrp3 x gut microbiota interactions on experimental colitis and insulin sensitivity

Felix Rost1, P. Pavlov1, S. Wueest2, K. Atrott1, G. Rogler1,3, D. Konrad2,3, I. Frey‐Wagner1,3

1Division of Gastroenterology and Hepatology, University Hospital Zurich, Raemistrasse 100, CH‐8091

Zurich, Switzerland; 2Division of Endocrinology and Diabetology, University Children’s Hospital Zurich,

Steinwiesstrasse 75, CH‐8032 Zurich Zurich, Switzerland; 3Zurich Center for Integrative Human Physiology, University of Zurich, Winterthurerstrasse 190, CH‐

8057 Zurich, Switzerland

[email protected]

Introduction: NOD‐like receptor pyrin domain containing 3 (Nlrp3) genotype has been shown to affect

murine experimental colitis and glucose metabolism. We here investigate whether Nlrp3 genotype‐

induced changes in gut microbiota composition are involved in the observed effects.

Methods: Female Nlrp3‐/‐ and wild‐type (WT) littermates underwent dextran sodium sulphate (DSS)‐

induced acute (7 days, 1% DSS) or chronic colitis (4 x 7 days, 1% DSS). Colitis severity was evaluated by

inflammatory parameters. Male Nlrp3‐/‐ and WT littermates received a high fat diet (HFD) or normal

chow for 6 weeks and glucose tolerance was assessed. Gut microbiota composition was studied by 16S

rRNA amplicon sequencing before and after induction of colitis / HFD.

Results: Nlrp3‐/‐ mice showed a more severe phenotype in acute and chronic DSS colitis. HFD‐fed

Nlrp3‐/‐ mice showed slightly improved glucose tolerance compared to WT littermates. Notably, Nlrp3‐

/‐ and corresponding WT littermates differed strongly in their gut microbiota composition under basal

conditions as well as after induction of colitis.

Discussion / Outlook: Gut microbiota composition, experimental colitis and glucose tolerance are

affected by the Nlrp3‐/‐ genotype. Further experiments with microbiota transfer between genotypes,

and colitis models / HFD experiments in animals with a limited defined flora will address a causal

relationship between gut microbiota composition and colitis / glucose tolerance outcome. Samples of

the Swiss IBD cohort (SIBDC) from patients with NLRP3 polymorphisms will be analyzed to compare

and extend the relevance of our findings to microbiota x genotype interactions in humans.


48

The role of HDAC6/Ubiquitin/aggresome pathway in the influenza virus infection

Alina Rudnicka, Yohei Yamauchi

University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse 190, CH‐8057 Zurich,

Switzerland

[email protected]

All viruses rely on host factors to support their life cycle. Identification and understanding of these

factors can facilitate development of antiviral drugs. Influenza A virus (IAV) is an enveloped single‐

stranded, negative‐sense RNA virus that belongs to the Orthomyxoviridae family. After its endocytic

uptake, infection of IAV depends on uncoating of the genome, protected in the virion by the M1 capsid

shell. Cytoplasmic histone deacetylase 6 (HDAC6) is a host factor that supports IAV capsid disassembly.

HDAC6 contains a zinc‐finger ubiquitin binding (ZnF‐UBP) domain that recognises unanchored

ubiquitin chains, a hallmark of misfolded protein aggregates. By packaging unanchored ubiquitin IAV

mimics a protein aggregate and hijacks HDAC6 to connect the capsid to motor proteins.

The goal of the project is to determine how HDAC6/Ubiquitin/aggresome pathway promotes IAV entry

and infection. Diverse approaches will be employed to interfere with HDAC6‐ubiquitin binding

followed by the analysis of their effects on IAV entry. First, IAV entry in MEF WT and HDAC6 KO cell

lines is compared by transmission electron microscopy. Second, we will target the HDAC6 ZnF‐UBP

domain by expressing nanobodies in A549 (human lung carcinoma) cells, and analyze their effect on

IAV uncoating and infection. Third, a targeted siRNA screen on ubiquitin‐related genes will be

performed in order to identify additional host factors that are necessary for IAV. This project is relevant

for future treatment strategies against viruses that utilize HDAC6 and aggresome processing.


49

Combining antigen formulations for human vaccination against cancer

Julia Rühl, Carol Leung, Christian Münz

University of Zurich, Institute of Experimental Immunology, Winterthurerstrasse 190, CH‐8057 Zürich

[email protected]

Epstein‐Barr virus (EBV) is a γ‐herpesvirus that preferentially infects B cells and establishes life‐long

chronic infection in more than 90% of the adult human population worldwide. The infection is usually

asymptomatic, and T cell responses clearly play an important role in maintaining the virus‐host

balance. However, when T cell immune control fails, EBV is associated with a number of human

malignancies such as Burkitt’s lymphoma, Hodgkin’s lymphoma and nasopharyngeal carcinoma. All

EBV‐positive tumor cells express the EBV nuclear antigen 1 (EBNA1), therefore EBNA1 is an important

target for protective immunity.

EBV has an exclusive tropism for humans, therefore the studied model organism is NOD‐‐/‐ mice with

human immune system components, which develop in these mice after neonatal intrahepatic injection

of human CD34+ hematopoietic progenitor cells (huNSG mice).

Targeting antigens to dendritic cells (DCs) is a promising strategy for therapeutic vaccinations. DCs are

important for initiating protective innate and adaptive immune responses against pathogens. T cell

responses can be enhanced through antigen delivery to endocytic receptors on DCs. Our group has

previously demonstrated that targeting EBNA1 to DEC‐205 elicits promising Th1‐type CD4+ T cell

responses in vitro and in vivo with a suitable adjuvant. However, targeting EBNA1 to DEC‐205 in vivo

induced poor CD8+ T cell responses. Therefore, we aim to improve the T cell responses by targeting

EBNA1 to different DC specific receptors in combination with other vector‐based delivery such as viral

vectors.


50

MCMV‐Specific T Cell Immunity in the Salivary Gland

Katharina Schmidt, Jenny Thom, Annette Oxenius

ETH Zurich – Institute of Microbiology, Vladimir‐Prelog‐Weg 4, CH‐8093 Zurich, Switzerland

[email protected]

Cytomegaloviruses (CMVs) are β‐herpesviruses infecting 60–90% of the human population. Even

though acute CMV infection is mostly asymptomatic in immunocompetent individuals, it can cause

fatal disease in the immunocompromised. Latency of the virus is established in secretory glands, such

as the salivary gland (SG), from where it continuously disseminates via mucosal secretions. Infection

of mice with murine CMV (MCMV) is a commonly used model to study the biology of infection.

MCMV infects many cells types but during established infection it mainly infects and replicates in acinar

glandular epithelial cells (AGECs) for several weeks in the SG. Prolonged infection in the SG is caused

by a failure of CD8+ T cells to control viral replication due to a virus‐mediated interference with

antigen‐presentation in infected AGECs. Therefore, IFNγ‐producing CD4+ T cells are required to

effectively control lytic infection at this site. Our group showed that systemic MCMV infection induces

tissue‐resident memory T cells (TRM) in the SG, either in an antigen‐dependent (CD4+) or –

independent (CD8+) manner. In striking contrast to CD8+ effector T cells during acute infection, we

showed that CD8+ TRM are able to mediate immediate protection against localized MCMV infection

of the SG. Here, we aim to characterize tissue intrinsic and viral factors responsible for the

susceptibility of the SG to MCMV infection. Additionally, we intend to use traceable MCMV‐specific T

cells in combination with GFP‐expressing MCMV to analyze the dynamic behavior of virally infected

cells and virus‐specific T cells in the SG by intravital microscopy.


51

Sickness behavior syndrome and clock gene disregulation in inflammatory bowel disease

Sellés‐Moreno C., Strauss L. & Fontana A.

Institute of Experimental Immunology, Winterthurerstrasse, 190 Y44 J68 Zürich 8003, Switzerland

[email protected]

Sickness behavior syndrome (SBS) as characterized by fatigue, depression and weight loss impairs

significantly the quality of life of patients with chronic inflammatory diseases. This holds also true for

an experimental model of inflammatory bowel disease (IBD), which is induced in mice with orally

administrated dextran sodium sulphate (DSS). The change of behavior becomes evident by loss of

locomotor activity as well as body weight loss. SBS is associated with altered clock gene expression in

the spleen and colon of mice with DSS colitis. Dysregulated clock gene expression has been shown to

be due to the effect of the

proinflammatory cytokines TNF‐a and IL‐1b.

We hypothesize that inflammation leading to the abnormal clock gene expression observed may

disrupt circadian rhythms resulting in fatigue, depression and metabolic disorders. Our new data show

that TNF and IL‐1β are largely produced by myeloid cells in inflamed tissue and blood. These cytokines

suppress the expression of clock output genes (Cry1 and 2, Per 1, Per2 and Per3 as well as the PAR bZip

transcription factors Dbp, Tef, Hlf) in vivo, and nuclear receptors involved in circadian rhythms

regulation .We find nuclear receptors involved in metabolic sensing are expressed in inflammation‐

activated macrophages and are directly implicated in controlling myeloid precursor commitment and

functional polarization. Preliminary results of our group show that expression patterns of clock and

nuclear receptors in inflamed colon and spleen in DSS‐mediated colitis in mice directly correlate with

body weight loss, disease severity and survival.


52

Characterization of Legionella effector proteins involved in the modulation of small GTPases

Leoni Swart, & Hubert Hilbi

University of Zürich, Institute of Medical Microbiology, Gloriastrasse 30/32, CH‐8006 Zürich, Switzerland

[email protected]

Legionella pneumophila is a ubiquitous water‐borne bacterium and the causative agent of the

severe pneumonia termed Legionnaires’ disease. The opportunistic pathogen infects lung

macrophages upon inhalation of contaminated aerosols. Once intracellular, the bacterium injects

approximately 300 effector proteins into the host cell via an Icm/Dot type IV secretion system (T4SS).

These proteins manipulate various host cell processes, like protein and vesicular trafficking, and

thereby prevent degradation of the pathogen via the bactericidal endolysosomal pathway. So far, only

a limited number of effectors have been characterized in detail.

Proteomic analysis of purified intact Legionella‐containing vacuoles (LCVs) revealed the presence

of hundreds of host proteins including a number of small GTPases implicated in the secretory and

endosomal vesicle trafficking pathway, as well as Ran and its effector Ran binding protein 1 (RanBP1).

The bacterial effector protein LegG1 has been found to activate Ran and thereby promote LCV

formation and motility, microtubule polymerization and bacterial replication.

Several members of the Rab GTPase family are also targeted by Legionella. Especially Rab1, which

is activated, inactivated and modified by bacterial effector proteins to regulate its function. AnkX has

been identified as a Legionella substrate which phosphocholinates Rab1 and thereby prevents the

inactivation of Rab1, yet also impairs binding downstream effector proteins. Hitherto, the exact

mechanism of action of most effector proteins, including LegG1 and AnkX, remains unknown. This PhD

project focuses on the characterization of LegG1 and its paralogues and orthologues, in particular the

mechanism of Ran activation. In addition, host targets and the phosphocholination mechanism of AnkX

will be investigated.


53

Caulobacter crescentus cell division is rate‐limited by peptidoglycan remodeling

Seamus Holden, Ambroise Lambert, Aster Vanhecke, Suliana Manley

École Polytechnique Féderale de Lausanne (EPFL), Laboratory of experimental biophysics (LEB), 1015 Lausanne, Switzerland

[email protected]

Bacterial cell division is a fundamental process central to bacterial growth and thus survival, involving

many highly conserved and essential genes. FtsZ is the first protein that is recruited to midcell, where

it forms a ring‐like structure and recruits a collection of proteins, termed the divisome, of which a lot

are involved in the remodeling of the cell wall. Despite meticulous research it is not known how and

to which degree the FtsZ‐ring and cell wall remodeling contribute to constriction. Using super‐

resolution microscopy, we were able to accurately measure the shape of Caulobacter crescentus cells

and thus also the size of the constriction site. High throughput imaging of synchronized cells provided

us with temporal information, which allowed us to measure the rate of constriction. We observe that

constriction, as measured by decrease in diameter, accelerates, such that the amount of newly

produced cell wall per unit time stays constant during constriction. Moreover, perturbations to various

targets involved in septal cell wall remodeling influence the constriction rate. We propose a model

where cell wall remodeling rate determines the rate of constriction.


54

Role of B cells in human rhinovirus infection

O.F. Wirz1, W. van der Veen1, C. Altunbulakli1, H. Morita1, S.L. Johnston2, N. Glanville2, C.A. Akdis1, and M. Akdis1

1SIAF, University of Zurich, Obere Strasse 22, Davos Platz, Switzerland; 2 Airway Disease Infection Section, National Heart and Lung Institute, Imperial College London, United Kingdom

[email protected]

Here, we studied the effect of infections of one major group rhinovirus (HRV 16) and one minor group

rhinovirus (HRV 29) on human immune system cells to investigate their roles in allergy and asthma

exacerbations and chronicity.

When PBMC from allergic and nonallergic individuals were stimulated with different types of HRVs, a

virus dose‐related cellular proliferation was observed, which was particularly confined to the B cell

compartment. We also found that CD19+ B‐cells could be infected by HRV16 and 29 in vitro. We

developed a set of primers to detect positive and negative strand viral RNA of HRV 16 and HRV 29.

While positive strand RNA will be detected from virus which is inside the cell as well as virus attached

to the cell surface, negative strand viral RNA can only be found when virus is in the replication process.

In our experiments, we could detect viral RNA in B cells at different timepoints after infection. Also,

pure B cells were infected with HRV16 and 29 and viral replication was also shown here. We could also

show that proliferating B cells, expressing elevated levels of ICAM‐1 show much smaller viral loads

than non‐proliferating B cells.

Our findings demonstrate for the first time, that human rhinoviruses have the ability to stimulate B

cells and activate plasmablasts. Furthermore, this study delivers the first evidence that B cells can be

infected by human rhinovirus.


55

HIV‐1 Integration Site Patterns in Monocyte‐derived Macrophages and CD4+ T Cells are “Same Same but Different”

Yik Lim Kok1,2, Valentina Vongrad1,2, Mohaned Shilaih1,2, Francesca D. Giallonardo3, Roger Kouyos1,2, Huldrych F. Günthard1,2, Karin J. Metzner1,2.

1Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, Zurich, Switzerland; 2Institute of Medical Virology, University of Zurich, Zurich, Switzerland; 3Marie Bashir Institute for Infectious Diseases and Biosecurity, Charles Perkins Centre, School of Biological Sciences and Sydney Medical School, The University of Sydney, Sydney, Australia.

[email protected]

The host’s genetic landscape surrounding integrated HIV‐1 has an impact on the fate of the provirus,

i.e. productive or latent. Studies analysing HIV‐1 integration sites in CD4+ T cells are numerous but

scarce for macrophages, the other major cellular target of HIV‐1. Here, we characterized HIV‐1

integration site patterns between monocyte‐derived macrophages (MDMs) and activated CD4+ T cells

within the same experimental settings. We analysed a total of 1,484 unique HIV‐1 integration sites

derived from seven ART‐treated HIV‐1‐infected individuals whose MDMs and CD4+ T cells were

isolated and infected ex vivo with autologous primary HIV‐1 isolates and heterologous HIV‐1. HIV‐1

integration site distribution in the human genome, basic host’s genetic requirements for HIV‐1

integration, and nucleotide selection specificity at HIV‐1 integration sites conferred by naturally

occurring amino acid polymorphisms in HIV‐1 integrase (S119P and T122I) were indistinguishable

between MDMs and CD4+ T cells. However, the repertoire of structural and functional HIV‐1 hosting

gene clusters in MDMs was distinct from that of CD4+ T cells. This distinction was further reinforced

by the different frequencies of HIV‐1 integration events in genes encoding HIV‐1 interacting proteins,

as well as frequencies of clonally expanded HIV‐1 hosting genes in gene hotspots between the two cell

types. These data suggest that different shock‐and‐kill strategies may be required to purge the entire

viral reservoir, and clonally expanded HIV‐1 hosting genes may be propagated by factors other than

the functional nature of the targeted genes.


56

Dissecting the biosynthetic pathway of O‐methylated glycans in Caenorhabditis elegans

Christina Zach, Markus Künzler, Markus Aebi

Vladimir‐Prelog‐Weg 1‐5/10, 8093 Zurich, Switzerland

[email protected]

Multicellular fungi produce cytoplasmic lectins implicated in fungal defense against predators.

Recently Tectonin 2, a lectin from the mushroom Laccaria bicolor, was proven toxic for the model

nematode Caenorhabditis elegans, by binding to nematode specific O‐methylated mannose and fucose

residues in the intestine.

Glycan methylation is widely recognized for bacteria, but is also found in nematodes, snails as wells as

algae and plants, however has not been found on mammalian glycans so far. The biosynthetic pathway

of this specific sugar modification remains still unclear. For Arabidopsis it was shown that methylation

of glucuronic acid takes place in the Golgi, where the methyl group is transferred from the donor

substrate S‐adenosylmethionine to the hydroxyl group of the mature glycan. This donor substrate is

synthesized in the cytosol and has to be transported into the Golgi lumen.

During a forward genetic screen, a C. elegans mutant was identified that is resistant to Tectonin

mediated toxicity and does no longer carry methylated glycans. The mutated gene encodes a protein

of the major facilitator superfamiliy. We hypothesize that this gene product is a Golgi‐localized

transporter, providing the donor substrate required for glycan methylation.

The aim of this project is on the one hand to verify the transport function of this putative transporter

in an in vitro assay and on the other hand, to identify the methyltransferases responsible for producing

these target epitopes. Therefore, I'm expressing candidate genes in insect cells in order to test for

enzyme activity in an in vitro assay.


57

Poster session I (Bärlocher – Mannweiler)

1) Kevin Bärlocher

Role of retrograde transport and RidL for intracellular replication of Legionella

2) Katja Becker

Structural and functional analysis of lipoprotein glycosylation in fast‐ and slowgrowing

mycobacteria

3) Desiree Böck

In vivo imaging of Salmonella invasion by electron cryotomography

4) Nathalie Cornillie Expression and characterization of the Plasmodium falciparum & Entamoeba histolytica

Oligosaccharyltransferase

5) Hanna Ebner The evolution of N332 glycan site directed broadly neutralizing antibodies in HIV infection

6) Hyuliya Emurla Asymmetric cell division and barrier function in T cell fate determination

7) Jillianne Eyring Purification and characterization of the octameric oligosaccharyltransferase from

Saccharomyces cerevisiae

8) Susanna Fleurkens Glycoengineering in insect cells to produce carbohydrate‐based vaccines against

Haemonchus contortus

9) Begonia Fudrini Characterization of Mycobacterium smegmatis PafBC

10) Fanny Georgi Correlative Multi‐Modal Imaging for 3D Spheroid Analyses

11) Christoph Gericke The influence of Alzheimer’s Disease‐like amyloid pathology on the immune surveillance in

the brain

12) Lisa Gottschlich Signaling in the General Stress Response of Sphingomonas melonis Fr1

13) Johannes Hartl Dynamic 13C labeling reveals in vivo half‐life of organic coenzymes in exponentially growing

14) Rebecca Higgins Hunt for somatic mutations in Linear Localized Scleroderma


58

15) Mario Hupfeld From protein function to supermolecular structure: Listeria phage A511 baseplate structure

and identification of receptor binding proteins

16) Stephan Isringhausen Dynamics of the bone‐marrow microenvironment during viral infections

17) Ivan Branislav Conformational protection of antibody epitopes on native HIV‐1 envelope glycoprotein

trimmers

18) Martina Lardi Response to nitrogen limitation in Burkholderia phymatum and competition between β‐

rhizobia for legumes infection

19) Katharina Littringer Infection‐induced changes in regulatory T cells

20) Olga Mannweiler Investigation of factors influencing pC3 stability within the Burkholderia cepacia complex

Poster session II (Martini – Zach)

21) Francesca Martini Glyco‐conjugate vaccine against Dirofilaria immitis

22) Corina Mathew N‐linked glycosylation in the Endoplasmatic Reticulum and Golgi

23) Andrea Mauracher Identification of a Novel Genetic Cause of Hypereosinophilia

24) Herbert A. Mbunkah Molecular Epidemiology of Transmitted HIV‐1 Drug Resistance in Drug‐Naive and Newly Diagnosed Patients in Cameroon

25) Fabian Meyer

Methylotrophy ‐ From understanding to engineering

26) Maximilian C.A. Mittelviefhaus Fluidic Force Microscopy ‐ A Novel Technology for Single‐Cell Manipulation and

Micropatterning


59

27) Christina Müller An In vivo study of HIV virus distribution and HIV‐Env specific antibodies in order to eliminate latently infected cells

28) Alessio Mylonas Anti‐TNF promotes type I interferon‐driven psoriasis‐like skin inflammation

29) Nikolas Rakebrandt Infection‐Induced Alterations of CD4+ T Cell Responses upon Heterologous Challenge

30) Felix Rost Impact of Nlrp3 x gut microbiota interactions on experimental colitis and insulin sensitivity

31) Alina Rudnicka

The role of HDAC6/Ubiquitin/aggresome pathway in the influenza virus infection

32) Julia Rühl Combining antigen formulations for human vaccination against cancer

33) Katharina Schmidt MCMV‐Specific T Cell Immunity in the Salivary Gland

34) Carla Sellés‐Moreno

Sickness behavior syndrome and clock gene disregulation in inflammatory bowel disease

35) Leoni Swart

Characterization of Legionella effector proteins involved in the modulation of small

GTPases

36) Aster Vanhecke

Caulobacter crescentus cell division is rate‐limited by peptidoglycan remodeling

37) Oliver Wirz Role of B cells in human rhinovirus infection

38) Kok Yik Lim HIV‐1 Integration Site Patterns in Monocyte‐derived Macrophages and CD4+ T Cells are

“Same Same but Different”

39) Christina Zach Dissecting the biosynthetic pathway of O‐methylated glycans in Caenorhabditis elegans


60

Participant details

Name Institute Email

Weronika Barcik SIAF [email protected]

Kevin Bärlocher Institute of Medical Microbiology [email protected]

Isabel Barnstorf Institute of Microbiology [email protected]

Katja Becker Medical Microbiology [email protected]

Desiree Böck Institute for Molecular Biology and Biophysics [email protected]

Nathalie Cornillie Institute of Microbiology [email protected]

Michel Crameri Institute of Medical Virology [email protected]

Hanna Ebner Institute of Medical Virology [email protected]

Hyuliya Emurla Institute of Microbiology and Immunology [email protected]

Jillianne Eyring Institute of Microbiology [email protected]

Susanna Fleurkens Institute of Microbiology [email protected]

Begonia Fudrini Institute of Medical Microbiology [email protected]

Fanny Georgi Institute of Molecular Life Sciences [email protected]

Christoph Gericke Division of Psychiatry Research [email protected]

Lisa Gottschlich Institute of Microbiology lisa‐[email protected]

Johannes Hartl Institute of Microbiology [email protected]

Rebecca Higgins University Hospital [email protected]

Mario Hupfeld INFH [email protected]

Stephan Isringhausen Experimental Hematology [email protected]

Ivan Branislav Institute of Medical Virology [email protected]

Corinna Landig Institute of Microbiology [email protected]

Martina Lardi Plant Biology [email protected]

Dagmara Lewandowska Institute of Medical Virology [email protected]

Katharina Littringer Institute of Experimental Immunology [email protected]

Monica Loi Institute of Experimental Immunology [email protected]

Olga Mannweiler Institute of Plant Biology [email protected]


61

Name Institute Email

Francesca Martini Institute of Microbiology [email protected]

Corina Mathew Institute of Microbiology [email protected]

Andrea Mauracher Paediatric Immunology Children's Hospital Zürich [email protected]

Herbert Afegenwi Mbunkah Division of Infectious Diseases and Hospital Epidemiology

[email protected]

Fabian Meyer Institute of Microbiology [email protected]

Max Mittelviefhaus Institute of Microbiology [email protected]

Christina Müller Division of Infectious Diseases and Hospital Epidemiology

[email protected]

Alessio Mylonas CHUV Dermatology [email protected]

Nikolas Rakebrandt Experimental Immunology [email protected]

Felix Rost Gastroenterology and Hepatology Unit [email protected]

Alina Rudnicka Institute of Molecular Life Sciences [email protected]

Julia Rühl Experimental Immunology [email protected]

Katharina Schmidt Institute of Microbiology [email protected]

Franziska Schönherr Virology [email protected]

Carla Sellés Moreno Institute of Experimental Immunology [email protected]

Bernhard Steiner IMM [email protected]

Emanuel Stiegeler IMV [email protected]

Leoni Swart Medical microbiology [email protected]

Aster Vanhecke EPFL [email protected]

Oliver Wirz SIAF [email protected]

Kok Yik Lim Division of Infectious Diseases & Hospital Edidemiology

[email protected]

Christina Zach Institute of Microbiology [email protected]

mim phd program - eth z · welcome to the 8th student retreat of the mim phd program dear...

Documents