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Institut de Biologie Moléculaire et Cellulaire CNRS UNIVERSITE DE STRASBOURG

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Niveau: Supérieur, Doctorat, Bac+8
Institut de Biologie Moléculaire et Cellulaire CNRS – UNIVERSITE DE STRASBOURG THESE DE DOCTORAT Discipline: Sciences du vivant Aspects Moléculaires et Cellulaires de la Biologie En vue d'obtenir le grade de Docteur de l'Université de Strasbourg Stefanie LIMMER Etude des relations hôte-pathogène dans des modèles d'infection intestinales de Drosophila melanogaster Soutenue le 19 Octobre 2010 devant la commission d'examen: Prof. Arturo ZYCHLINSKY (Rapporteur Externe), Max Planck Institut für Infektionsbiologie, Berlin Dr. Jonathan EWBANK (Rapporteur Externe), U631 de l'INSERM, Centre d'Immunologie, Marseille Prof. Philippe GEORGEL (Examinateur), Laboratoire d'Immunogénétique Moléculaire, Strasbourg Prof. Jules HOFFMANN (Examinateur), UPR9022 du CNRS, Strasbourg Prof. Christian KLÄMBT (Examinateur), Institut für Neurobiologie, Münster Dr. Dominique FERRANDON (Directeur de thèse), UPR9022 du CNRS, Strasbourg

  • spz spätzle

  • diap2 drosophila

  • c4-hsl butanoyl-homoserine lactone

  • modèles d'infection intestinales de drosophila melanogaster

  • mitogen activated protein

  • drosophila melanogaster

  • spe spätzle-processing-enzyme

  • kinase

  • enzyme pqs


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Published 01 October 2010
Reads 18
Language English
Document size 9 MB

Institut de Biologie Moléculaire et Cellulaire
CNRS – UNIVERSITE DE STRASBOURG




THESE DE DOCTORAT
Discipline: Sciences du vivant
Aspects Moléculaires et Cellulaires de la Biologie

En vue d’obtenir le grade de
Docteur de l’Université de Strasbourg



Stefanie LIMMER



Etude des relations hôte-pathogène dans des modèles
d’infection intestinales de Drosophila melanogaster





Soutenue le 19 Octobre 2010 devant la commission d’examen:

Prof. Arturo ZYCHLINSKY (Rapporteur Externe), Max Planck Institut für Infektionsbiologie, Berlin
Dr. Jonathan EWBANK (Rapporteur Externe), U631 de l’INSERM, Centre d'Immunologie, Marseille
Prof. Philippe GEORGEL (Examinateur), Laboratoire d’Immunogénétique Moléculaire, Strasbourg
Prof. Jules HOFFMANN (Examinateur), UPR9022 du CNRS, Strasbourg
Prof. Christian KLÄMBT (Examinateur), Institut für Neurobiologie, Münster
Dr. Dominique FERRANDON (Directeur de thèse), UPR9022 du CNRS, Strasbourg
Acknowledgment

First, I want to thank Prof. Jules Hoffmann and Prof. Jean-Marc Reichhart for having
made the laboratory one of the best in the field of immunology and a fruitful
environment for young researchers.

I also want to thank Prof. Arturo Zychlinsky, Dr. Jonathan Ewbank, Prof. Philippe
Georgel, Prof. Jules Hoffmann and Prof. Christian Klämbt for taking the time to read
and judge my work.

Dominique, over all the years you were always there to help and encourage me. You
also took the time for many, many fruitful discussions. I’m really happy that I had the
possibility to work in your group. I learned a lot during my time in Strasbourg. Thank
you.

Nadine, thank you for establishing the Serratia model and for introducing me into the
subject.

A big thanks to Cordu, Ioannis, Safia, Basti, Magda, Stan, Jessica, Ayyaz and Sunny
for their friendship and help. I’ll miss you all. Steffi, what should I say? Thank you! I
also want to thank all the other current and former members of the team and the rest
of the lab for all their help and support. Samantha: good luck for your thesis.

Last but not least, I want to thank my family and my boyfriend for their support and
encouragement over the years. Without you I would not have managed. Thank you. Abbreviations

3-oxo-C -HSL 3-oxo-dodecanoyl-homoserine lactone 12
AMP anti-microbial peptide
C -HSL butanoyl-homoserine lactone 4
CF cystic fibrosis
DAP diaminopimelic acid
DIAP2 Drosophila inhibitor of apoptosis 2
Dif Dorsal related immunity factor
Dome Domeless
Dscam Down syndrome cell adhesion molecule
DUOX dual oxidase
EB enteroblast
EC enterocyte
EMS ethyl methanesulfonate
Erk extracellular signal-regulated kinase
GlcNAc N-acetylglucosamine
GNBP gram-negative binding protein
GPCR G-protein coupled receptor
Gprk G-protein-coupled receptor kinase
hFAF1 human Fas associated factor 1
Hop Hopscotch
IAP inhibitor of apoptosis
IBM IAP-binding motif
IMD Immune deficiency
IP3 1,4,5-triphosphate
IRAK IL-1R associated kinase
ISC intestinal stem cell
Key Kenny
LPS lipopolysaccharide
LRR leucine-rich repeats
Lys lysine
MAMP microbe associated molecular pattern
MAPK mitogen-activated protein kinase ModSP modular serine protease
MurNAc N-acetylmuramic acid
N-acyl-HSL N-acylhomoserine lactone
NADPH nicotineamide adenine dinucleotide phosphate
ORF open reading frame
PGN peptidoglycan
PGRPs PGN-recognition proteins
PLCβ phospholipase C-β
PO phenoloxidase
PPAE Prophenoloxidase activating enzyme
PQS Pseudomonas Quinolone Signal
ProPO prophenoloxidase
PRRs pattern recognition receptors
Psh Persephone
Pvf PDGF- and VEGF-related factor
Pvr PDGF- and VEGF-receptor related
QS quorum sensing
ROS reactive oxygen species
ROS reactive oxygen species
SPE Spätzle-processing-enzyme
Spz Spätzle
Tep Thioester-containing protein
TLR Toll-like receptor
Tot Turandot
TPSS two partner secretion system
upd unpaired
WntD wnt inhibitor of Dorsal
βGRP β-glucan recognition proteins
Content 1
1 Introduction.......................................................................................................... 3
1.1 Overview ...................................................................................................... 4
1.2 Drosophila melanogaster ............................................................................. 5
Systemic response .............................................................................................. 6
Recognition of microbes .................................................................................. 6
Recognition of Gram(-) bacteria via DAP-type Peptidoglycan (PGN)........... 7
Recognition of Gram(+) bacteria via Lys-type Peptidoglycan (PGN).......... 10
Recognition of fungi.................................................................................... 11
Signal transduction ........................................................................................ 12
Activation of the Toll pathway..................................................................... 12
The Toll pathway........................................................................................ 13
Negative regulation of the Toll pathway ..................................................... 15
The IMD pathway ....................................................................................... 15
Negative regulation of the IMD pathway..................................................... 17
The JAK/STAT pathway ............................................................................. 21
Immune effectors ........................................................................................... 24
Antimicrobial peptides ................................................................................ 24
Tep proteins ............................................................................................... 24
Other effectors............................................................................................ 25
Local immune responses................................................................................... 25
Physical barrier and hostile environment in the midgut .............................. 26
AMP expression ......................................................................................... 27
ROS production.......................................................................................... 28
Cellular immune response................................................................................. 29
Phagocytosis.............................................................................................. 30
Encapsulation............................................................................................. 30
Coagulation ................................................................................................ 31
Melanization ............................................................................................... 32
Other immune functions of hemocyte......................................................... 32
1.3 Serratia marcescens .................................................................................. 34
The bacterium ............................................................................................ 34
S. marcescens infection in Drosophila ....................................................... 36
1.4 Pseudomonas aeruginosa.......................................................................... 37
The bacterium ............................................................................................ 37
Content 2
P. aeruginosa infections in Drosophila ....................................................... 41
1.5 Aim of this work.......................................................................................... 41
2 Serratia marcescens infections ......................................................................... 43
2.1 Genome-Wide RNAi screen identifies genes involved in intestinal
pathogenic bacterial infection................................................................................ 44
Introduction........................................................................................................ 44
Additional results and discussion ...................................................................... 47
Validation of candidate genes .................................................................... 47
JAK/STAT pathway and compensatory proliferation .................................. 52
2.2 Six hour-long regeneration of the Drosophila melanogaster midgut following
its partial degradation by ingested Serratia marcescens....................................... 57
Introduction........................................................................................................ 57
Discussion ......................................................................................................... 59
3 Pseudomonas aeruginosa infections................................................................. 61
3.1 Pseudomonas aeruginosa RhlR is required to neutralize the cellular immune
response in a Drosophila melanogaster oral infection model................................ 62
Introduction........................................................................................................ 62
Further characterization of the oral infection by P. aeruginosa and discussion. 67
How does P. aeruginosa manage to cross the gut epithelium?.................. 67
What triggers a switch to virulence in the hemolymph?.............................. 69
Are the host defense responses independent of each other and if yes, how
does P. aeruginosa trigger the Toll pathway? ............................................ 71
Is the role of RhlR during infection quorum sensing dependent? ............... 76
What is the role of the T2SS? .................................................................... 79
4 Concluding remarks........................................................................................... 82
The model system...................................................................................... 83
Intestinal infections..................................................................................... 84
Virulence in the hemocoel .......................................................................... 85
Tolerance/endurance, an important mechanism of host resistance ........... 85
5 Annex ................................................................................................................ 88
6 Bibliography....................................................................................................... 90



Introduction 3















1 Introduction
Introduction 4
1.1 Overview
Multicellular organisms have to face life-challenging infection by a variety of microbes
over and over. Therefore, throughout the evolution, the animal and plant phyla
developed powerful mechanisms to fight invading microorganisms. Being able to
sense different microbes and to induce appropriate defenses, which means having a
potent immune system is a key advantage to host survival. These basic defense
mechanisms appeared early in the evolution of multicellular organisms and are
referred to as innate immunity. The innate immune system involves germ-line
encoded receptors that are able to recognize infectious non-self particles and
subsequently trigger the expression of effectors that target the microorganisms. Later
in evolution, in the ancestors of cartilaginous fish, another arm of immunity appeared:
adaptive immunity. It is restricted to vertebrates and displays a second line of
defense in addition to the innate immune system. The adaptive immune system relies
on the generation of a complex repertoire of immune receptors in lymphocytes. This
huge variety of receptors is generated by somatic gene rearrangement. Innate
immunity reactions trigger the adaptive immune response and orient the effector
mechanisms of this response (Fearon 1997; Janeway et al. 2002). In addition to
fighting invading microorganism, it is essential for the host to be able to deal with
damage caused by the microbes.
Pathogenic microorganisms have coevolved with their hosts, always
developing novel strategies to overcome the defense mechanisms of multicellular
organisms. The first barriers microbes face are physical, like skin or cuticle and
barrier epithelia in, for example, respiratory or digestive organs. After having
overcome these barriers, they have to withstand the attack of the immune system to
successfully infect the host. For this purpose they have developed sophisticated
strategies and weaponry. To date, we by far do not understand all interactions
between host and pathogen that lead to infectious diseases.

Therefore, the goal of my PhD was to use the strength of genetics to better
understand host-pathogen interactions between the genetic model organism
Drosophila melanogaster and two Gram(-) bacteria, Serratia marcescens and
Pseudomonas aeruginosa, in an oral infection model.
Introduction 5
1.2 Drosophila melanogaster
The fruit fly as a model organism has several advantages. It is very small and easy to
maintain. The short life cycle and its high number of offspring allow to obtain high
numbers of flies and permit fast genetic manipulation. Furthermore, a century of
working with Drosophila generated many powerful genetic tools. The genome of
Drosophila has been fully sequenced (Adams et al. 2000) and large collections of
mutant and transgenic lines are accessible. The yeast UAS-GAL4 system is widely
used in Drosophila to generate transgenic lines, in which transgene expression can
be induced in a spatio-temporally controlled manner (Brand et al. 1993). In addition,
saturation mutagenesis is achievable by using several techniques. Chemical
mutagenesis by feeding ethyl methanesulfonate (EMS) to the flies, for example,
creates point mutations (Jenkins 1967). Transposon-mediated mutagenesis, which
leads to a disruption or deregulation of gene expression (Rubin et al. 1982), is
another possibility. A method to downregulate gene expression in an inducible
manner is to combine the UAS-GAL4 system with RNA interference (RNAi), by
expressing a hairpin dsRNA construct, targeting the gene of interest, under the
control of a GAL4 promoter (Kennerdell et al. 2000). In addition to diverse genetic
tools such as balancer chromosomes, this palette of mutagenesis techniques renders
Drosophila a powerful genetic model.
As 80% of extant organisms Drosophila is highly resistant to microbial infection, even
though it has no adaptive immune system and therefore completely relies on the
innate immune response. This makes the immune system of the fly easier to study
since the adaptive arm of the immune system cannot mask the phenotypic effects of
mutations in genes implicated in innate immunity. Therefore it is easier to correlate a
genetic mutation to a phenotype. The immune system of the fly consists of several
mechanisms. After wounding, several proteolytic cascades are activated, of which
one leads to the deposition of melanin at the wound site and the production of
cytotoxic reactive oxygen species (ROS) that antagonize invading microorganisms
(Nappi et al. 1993). Those microbes are also dealt with by hemocytes, which are
capable of phagocyting invaders (Braun et al. 1998). Injury as well as the presence of
microbes in the hemocoel leads to the systemic induction of antimicrobial gene
expression in the fat body, a functional equivalent of the mammalian liver. Sensing of
wounding or invading microbes triggers many genes including those coding for
antimicrobial peptides (AMPs) via, amongst others, two NF-κB pathways, the Toll and
Introduction 6
the Immune deficiency (IMD) pathway. AMPs are secreted into the hemolymph,
where they counteract the infection (reviewed in (Ferrandon et al. 2007; Lemaitre et
al. 2007), Figure 1). The epithelia of the fly provide the first barrier against
microorganisms both at the physical and chemical level by secreting AMPs and ROS
(Ferrandon et al. 1998; Ha et al. 2005a). In the following I shall introduce the
systemic, cellular, and epithelial immune responses of the fly in more detail.

Figure 1: The Drosophila systemic immune reaction. A septic wound triggers the systemic immune
reaction of the fly. The melanization and coagulation cascades are activated to trap pathogens and
close the wound. Invading microbes are phagocytosed by hemocytes. In addition, the production of
reactive oxygen intermediates might be triggered to fight microorganisms. A systemic infection, as well
as wounding to some extent, induces the IMD and Toll pathway-dependent production of antimicrobial
peptides (AMPs) by the fat body and their secretion into the hemolymph. From Limmer et al. (see
Annex)
Systemic response
Recognition of microbes
Since pathogenic microorganisms are very diverse, any living organism requires
several defense mechanisms to be able to fight efficiently different invaders. In order
to raise an appropriate reaction, the immune system needs the ability to distinguish
between distinct classes of microbes. The immune system of Drosophila has
developed several sensing mechanisms to differentiate between Gram(+) bacteria,