Thesis
Reference
Host-pathogen interactions: from bacterial virulence to host defense
LE COADIC, Marion
Abstract
La compréhension des mécanismes régissant les interactions entre un hôte et un pathogène est essentielle pour élaborer des stratégies thérapeutiques permettant une complète élimination du pathogène tout en minimisant les dommages causés à l'hôte. Une infection par un pathogène implique deux principaux paramètres: la capacité du pathogène à envahir l'hôte à l'aide de facteurs de virulence, et la capacité de l'hôte à se défendre grâce à son système immunitaire. Au cours de ma thèse j'ai pu étudier à la fois la virulence bactérienne et les mécanismes de défense de l'hôte. En effet, j'ai montré la possibilité d'utiliser la puce d'eau Daphnia magna comme modèle système pour évaluer la virulence de bactéries ; Et en utilisant l'amibe Dictyostelium discoideum comme modèle de cellule phagocytaire, j'ai pu clarifier le rôle des protéines SadA, Phg1A, SibA et Kil1 au cours d'étapes spécifiques du mécanisme de phagocytose.
LE COADIC, Marion. Host-pathogen interactions: from bacterial virulence to host defense. Thèse de doctorat : Univ. Genève, 2012, no. Sc. 4488
URN : urn:nbn:ch:unige-266829
DOI : 10.13097/archive-ouverte/unige:26682
Available at:
http://archive-ouverte.unige.ch/unige:26682
Disclaimer: layout of this document may differ from the published version.
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UNIVERSITÉ DE GENÈVE
Département de biologie cellulaire FACULTÉ DES SCIENCES Professeur Jean-Claude Martinou
Département de physiologie cellulaire FACULTE DE MÉDECINE
et métabolisme Professeur Pierre Cosson
Host-pathogen interactions: from bacterial virulence to host defense
THÈSE
présentée à la faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès science, mention biologie
par
Marion Le Coadic de
Lorient (France)
Thèse N°4488
Genève
Atelier de reprographie Repromail 2012
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“Il n’y a de sciences que dans la mesure où l’on plonge le réel dans un virtuel contrôlé”
René Thom Prédire n’est pas expliquer
“Face au réel, ce que l’on croit savoir clairement offusque ce que l’on devrait savoir. Quand il se présente à la culture scientifique, l’esprit n’est jamais jeune. Il est même très vieux, car il a l’âge de ses préjugés. Accéder à la science, c’est spirituellement rajeunir, c’est accepter une mutation brusque qui doit contredire un passé”
Gaston Bachelard La formation de l’esprit scientifique
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REMERCIEMENTS
Tout d’abord, je tiens à remercier Pierre pour m’avoir permis de former mon esprit scientifique à ses côtés. Ta passion pour la connaissance, ta rigueur intellectuelle et ton dynamisme auront été pour moi des exemples et des moteurs. Je continue de penser que l’important dans une thèse c’est son chef ! Merci
Je remercie les membres de mon jury, Pierre Golstein, François Letourneur et Jean-Claude Martinou pour avoir accepté d’évaluer mon travail.
Un grand merci à Emmanuelle ! Pour ton soutien bienveillant, ta gentillesse et tout ce que j’ai eu la chance de partager avec toi. Tu es ma merveilleuse amie. Vincent et toi, ainsi que Valérie, êtes ma famille d’ici.
Je remercie tous les membres du laboratoire avec qui j’ai partagé mes années de thèse: Jackie, Hajer, Cristiana, Marco, Anna, Wanessa, Romain, Cédric, Madeleine, Laura, ainsi que les nouveaux arrivants Alexandre et Jessica. C’était un vrai plaisir de vous retrouver tous les jours, et de partager des moments de détente, faits de rire et d’échanges. Vous me manquerez.
Merci à Priscilla, pour son amitié et nos discussions qui m’ont ouvert de nouveaux horizons. Merci aussi à Philippe pour m’avoir fait découvrir Entamoeba gingivalis !
Merci à tous les membres du département PHYME et en particulier à Olivier, Hervé, Tamara, Dominique, Annelise et Corine pour leur aide précieuse et leur patience. Merci aussi à Laurène, Christian, Marie-Claude, Cyril, Denis, Séverine, Perrine et Lelio, pour leur gentillesse et tous les bons moments passés en votre compagnie.
Merci à tous mes amis qui ont vécu de près ou de loin cette aventure.
Les derniers mais non les moindres :
Fred, mon compagnon du quotidien, qui a vécu les week-ends de travail, les retours tardifs du laboratoire, les doutes et qui n’a pas cessé de m’encourager. Te savoir à mes côtés aura été mon carburant. Grâce à toi je n’ai rien lâché !
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Merci ma sœur Mona, pour toutes nos discussions « philosophiques » ponctuées de grands moments de rire, pour nos road trips improvisés, pour cette belle énergie positive qui t’habite et ta capacité à penser en dehors du cadre. Tu es une belle source d’inspiration pour moi.
Merci ma sœur Maëlle, pour ton soutien moral, ta bienveillance envers moi, ton intérêt pour mon sujet de thèse même si je sais que je n’ai pas toujours été très claire. Avec Matéo et Sacha vous êtes une bouffée de vitalité et d’amour, qui me remplit d’énergie à chaque fois que je vous vois.
Enfin, je ne saurai exprimer en quelques lignes toute ma gratitude envers vous, mes chers parents.
Je dirai simplement que c’est sans aucun doute grâce à vous que j’ai pu vivre cette formidable expérience. Vous m’avez apporté le goût de l’exploration et l’envie d’apprendre. Et vous m’avez soutenue, encouragée, et entourée de votre amour tout au long de cette aventure que vous-même avez vécue. Merci !
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CONTENTS
FIGURES and TABLE p. 12
RESUME p. 14
SUMMARY p. 16
INTRODUCTION p. 18
I. HOST-PATHOGEN INTERACTIONS p. 18
I. A. Bacterial colonization: healthy versus pathogenic situation p. 18
I. A. 1. Resident microbiota and opportunistic pathogens p. 18
I. A. 2. True pathogenic bacteria and emergent pathogens p. 19
I. A. 3. Virulence factors of Pseudomonas sp. and Photorhabdus sp. p. 20
a. Adhesion systems p. 21
b. Two component system (TCS) and quorum sensing (QS) p. 25
c. Toxins p. 27
I. B. Measuring bacterial virulence p. 28
I. B. 1. Animal models to study bacterial virulence p. 28
a. Mus musculus p. 28
b. Danio rerio p. 29
c. Drosophila melanogaster p. 30
d. Caenorhabditis elegans p. 32
e. Arabidopsis thaliana p. 33
f. Dictyostelium discoideum p. 33
I. B. 2. Daphnia magna as a model system p. 37
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II. HOST DEFENSE p. 42
II. A. Generalities p. 42
II. A. 1. The mammalian immune system p. 42
a. Innate and adaptive immunity p. 42
b. Professional phagocytes p. 43
II. A. 2. Phagocytosis and membrane trafficking p. 43
II. A. 3. Objectives of my thesis p. 44
II. B. Adhesion of phagocytes to bacteria p. 46
II. B. 1. Adhesion of mammalian phagocytes to bacteria p. 46
a. Toll like receptors p. 46
b. Scavenger receptors p. 47
c. Opsono-receptors p. 47
II. B. 2. Receptors for phagocytosis in Dictyostelium p. 49
a. Phg1 p. 50
b. SadA p. 52
c. SibA p. 52
II. C. Internalization of bacteria and maturation of the phagosome p. 54
II. C. 1. Phagocytosis in mammalian phagocytes p. 54 a. Fc!Rs- and CR3-mediated phagocytosis p. 54
b. From phagosome to phagolysosome p. 56
II. C. 2. Bacterial uptake and phagosome maturation in Dictyostelium p. 58
II. D. Intracellular killing of bacteria p. 62
II. D. 1. Bacterial killing in the mammalian phagolysosome p. 62
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a. The respiratory burst p. 62
b. Acidification p. 63
c. Ions p. 64
d. Lysosomal enzymes p. 65
II. D. 2. Intracellular killing in Dictyostelium p. 67 a. The role of mammalian orthologs p. 67 b. New gene products implicated in killing p. 68
RESULTS p. 72
I. Results: publication p. 72
Daphnia magna, a host for evaluation of bacterial virulence
II. Results: publication p. 75
TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium
III. Results: Intracellular sorting of SibA depends on Phg1A p. 77
III. 1. Introduction p. 77
III. 2. Results p. 78
a. Localization of csA-SibA TM in phg1A KO cells p. 78 b. The secretion of csA, but not its stability, is impaired p. 80
in phg1A KO
c. Glycine residues in the SibA TMD are recognized in a p. 81 Phg1A-dependent manner
d. Phg1A interacts specifically with glycine-rich TMDs p. 82
III. 3. Discussion p. 82
III. 4. Materials and methods p. 85
III. 5. Figure legends p. 88
IV. Results: publication p. 103
Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium
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GENERAL DISCUSSION p. 106
ANNEX: publication p. 118
STIM1-induced precortical and cortical subdomains of the endoplasmic reticulum
BIBLIOGRAPHY p. 121
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FIGURES and TABLE
Figure 1: Bacterial adhesion molecules p. 23
Figure 2: Regulatory systems controlling expression of virulence factors p. 26 in P. aeruginosa
Figure 3: Phylogenetic tree indicating major model systems p. 32 Figure 4: Dictyostelium: from unicellularity to multicellularity p. 35 Figure 5: Assessing bacterial virulence by using Dictyostelium p. 36
Figure 6: Annotation of the Daphnia anatomy p. 39
Figure 7: Daphnia life cycle p. 40
Figure 8: Membrane trafficking p. 45
Figure 9: Interaction of PRRs with MAMPs p. 48
Figure 10: Phylogenetic tree of TM9SF proteins p. 51
Figure 11: Fc!R- and CR3-mediated phagocytosis p. 55
Figure 12: Phagosome maturation p. 57
Figure 13: Internalization of bacteria in Dictyostelium is an actin-based p. 60 mechanism similar to those in mammalian phagocytes
Figure 14: The phagocytic pathway in Dictyostelium p. 61
Figure 15: Bacterial killing in phagolysosome p. 66
Figure 16: Specific growth defect of phg1A KO, kil2 KO and kil1 KO cells p. 70 on K. aerogenes
Figure 17: Chimeric csA fusion proteins p. 88
Figure 18: Immunofluorescence labeling of csA p. 88
Figure 19: Detection of csA surface expression by flow cytometry p. 89 Figure 20: Analysis of cell surface localization of csA chimeras in WT and p. 90
phg1A KO cells by flow cytometry
Figure 21: Analysis of flow cytometry data p. 91
Figure 22: Surface localization of csA-SibA TM and csA-TM in WT and p. 92 phg1A KO cells
Figure 23: Cell surface localization of csA-SibA TM visualized by p. 93 immunofluorescence
Figure 24: Cell surface localization of csA analyzed by cell surface biotinylation p. 94 Figure 25: Surface and total csA analysis following cell surface biotinylation p. 95
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Figure 26: Mature csA exhibits two carbohydrates p. 96
Figure 27: csA-SibATM and csA-TM exhibit similar stability in WT and p. 97 phg1A KO cells
Figure 28: The SibA transmembrane domain exhibits a glycine motif p. 98 Figure 29: The cell surface localization of glycine-rich TM domains depends on p. 99
Phg1A
Figure 30: Chimeric Tac fusion proteins p. 100
Figure 31: Assessing interaction between Phg1A and Tac chimeric proteins p. 101 Figure 32: Phg1/TM9SF4 associates with glycine-rich transmembrane domains p. 102 Figure 33: Bacterial virulence in different model systems p. 108 Figure 34: Model of SibA-mediated adhesion controlled by Phg1A or SadA p. 111 Figure 35: Kil1 localizes in intracellular compartments that are not recycling p. 114
endosomes
Figure 36: Kil1 colocalizes with Golgi marker but not endoplasmic reticulum p. 115 marker
Table 1: Model systems to study bacterial virulence p. 41
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RÉSUMÉ
La compréhension des mécanismes régissant les interactions entre un hôte et un pathogène est essentielle pour élaborer des stratégies thérapeutiques permettant une complète élimination du pathogène tout en minimisant les dommages causés à l’hôte. Une infection par un pathogène implique deux principaux paramètres: la capacité du pathogène à envahir l’hôte à l’aide de facteurs de virulence, et la capacité de l’hôte à se défendre grâce à son système immunitaire.
Au cours de ma thèse j’ai pu étudier à la fois la virulence bactérienne et les mécanismes de défense de l’hôte.
L’évaluation de la virulence des bactéries isolées chez des patients est la première étape qui mène à l’identification de l’agent responsable de la maladie, et par la suite, qui permet d’identifier ce qui en particulier rend la bactérie pathogène, autrement dit d’identifier les facteurs de virulence de la bactérie. Aujourd’hui différents modèles animaux ainsi que des plantes et protistes sont utilisés pour évaluer la virulence des bactéries et pour découvrir de nouveaux aspects des mécanismes de la virulence bactérienne. Dans la première partie des résultats de ma thèse (Results I) je montre la possibilité d’utiliser la puce d’eau Daphnia magna comme modèle système pour évaluer la virulence de bactéries.
Pour étudier le deuxième aspect des interactions hôte-pathogène qui est la réponse de l’hôte lors d’une infection, j’ai utilisé l’amibe Dictyostelium discoideum comme modèle de cellule phagocytaire. En effet, cette amibe est un phagocyte professionnel qui se nourrit naturellement de bactéries et présente de fortes similarités avec les cellules phagocytaires humaines telles que les macrophages ou les neutrophiles. Ces dernières sont les premiers acteurs de la réponse immunitaire chez l’humain en cas d’invasion par un pathogène. Elles ont la capacité d’engouffrer et de tuer très efficacement les pathogènes, d’où leurs noms de cellules phagocytaires. Du fait de sa ressemblance avec les cellules phagocytaires humaines, de sa facilité de manipulation et des outils génétiques pratiques que Dictyostelium présente, cette amibe est un devenu un modèle privilégié pour l’étude de la phagocytose. Le mécanisme de la phagocytose peut être découpé en quatre principales étapes : l’adhésion de la cellule à la bactérie, l’internalisation de la bactérie dans un phagosome, la maturation du phagosome en phagolysosome et l’élimination des bactéries dans le phagolysosome.
Dans la seconde partie des résultats de ma thèse je traite essentiellement des mécanismes d’adhésion de la cellule à la bactérie et des mécanismes d’élimination dans le phagolysosome. Plus précisément je montre les rôles régulateurs des protéines SadA et Phg1A dans l’expression de surface de la molécule d’adhésion SibA (Results II), puis je montre plus spécifiquement que l’expression à la surface de la protéine SibA dépend de l’interaction du domaine transmembranaire de SibA avec Phg1A (Results III). Enfin dans une troisième étude je montre que la protéine Kil1 est
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essentielle à l’élimination de la bactérie Klebsiella aerogenes, et que l’expression de Kil1 est sous le contrôle de Phg1A/B. Par ailleurs je montre que Kil2, une protéine impliquée dans l’élimination de K. aerogenes, intervient dans un mécanisme distinct de celui de Phg1A et Kil1 (Results IV).
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SUMMARY
Understanding the genetic and molecular basis of the interactions between a host and a pathogen is essential to elaborate therapeutic strategies, ensuring a complete elimination of the pathogen while minimizing host damages. Infection by a pathogen depends on two main parameters: the ability of the pathogen to invade and cause damage to the host, provided by the virulence mechanisms of the pathogen, and the ability of the host to combat the pathogen, provided by the immune system of the host. During my PhD, I had the opportunity to study both bacterial virulence and host response mechanisms.
Evaluating bacterial virulence is essential to identify what particular features (virulence factors) make bacteria pathogenic. Today several animal models of infection exist, but plants and protists can also be used to measure bacterial virulence and to provide new insights on bacterial virulence mechanisms. In the first part of my results (Results I) I showed that the water flea Daphnia magna can be used as a model system to evaluate bacterial virulence and I describe its advantages:
numerous bacteria can be tested in the same experiment, results can be obtained in one day and it is a very low-cost model.
To investigate the second facet of host-pathogen interactions, i.e. the host response, I used the amoebae Dictyostelium discoideum as a professional phagocyte model. Indeed, D. discoideum feeds upon bacteria and resembles mammalian professional phagocytes such as macrophages and neutrophils. These latter are the first line of defense when pathogens invade the human organism.
They have the ability to engulf and kill bacteria very efficiently. Because of the similarities of Dictyostelium with mammalian phagocytes and because of its numerous advantages as a model system, this amoeba has become a reference model to study phagocytosis. Phagocytosis can be divided in four main steps: adhesion of the phagocyte to the bacteria, internalization of the bacteria in a phagosome, maturation of the phagosome into a phagolysosome and killing of bacteria in the phagolysosome. In the second part of my thesis I investigated the mechanisms controlling adhesion and bacterial killing in the phagolysosome. More specifically, I showed that Phg1A and SadA control the expression of SibA, an adhesion molecule, at the cell surface (Results II). I further showed that cell surface expression of SibA depends on the interaction of Phg1A with the transmembrane domain of SibA (Results III). Finally, in a third study (Results IV) I showed that the protein Kil1 is essential for the elimination of K. aerogenes and that the expression of Kil1 is controlled by Phg1A/B. In addition I showed that Kil2 participates in the killing of K. aerogenes through a distinct mechanism.
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INTRODUCTION
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INTRODUCTION
I. HOST-PATHOGEN INTERACTIONS
Bacteria are prokaryotic organisms colonizing almost every surface on earth including plants and animals (Grice et al., 2009; Sears, 2005; Whitman et al., 1998). Interactions with bacteria can be beneficial or deleterious. Bacteria that have evolved a mutualistic relationship with a host, providing beneficial effects both to the host and to bacteria, often form the resident microbiota (I. A.
1.). Bacteria causing a disease by damaging the host integrity are named pathogens (I. A. 2.), a term derived from the Greek words pathos “suffering” and gennan “producer of”. Pathogens trigger a disease in the host by the action of virulence factors that are described in the part I. A. 3. Animal models are used to evaluate and study bacterial virulence factors (I. B. 1.). During my PhD I aimed to investigate on the putative use of the water flea Daphnia magna (I. B. 2) as model system to evaluate the virulence of bacteria.
I. A. Bacterial colonization in mammals: healthy versus pathogenic situation
I. A.1 Resident microbiota and opportunistic pathogens.
Mutualistic relationships have emerged during evolution between some bacteria, named resident microbiota, and virtually every multicellular organism, such as human, fish, worms or plants. These interactions provide beneficial effects to both the bacteria, which find an appropriate niche to multiply, and the host. For example, resident bacteria in the human gut ensure the digestion of complex polysaccharides and the production of vitamins K and B12 (Resta, 2009). The mutualistic relationship of human gut microbiota has also the ability to stimulate the immune system of the host without triggering inflammation. A stimulated immune system makes the host more resistant to infections by pathogenic bacteria (Kelly et al., 2007; Rakoff-Nahoum et al., 2004; Wanke et al., 2011). Germ free mice have thus been shown to be particularly sensitive to infection by pathogens (Fagundes et al., 2012).
Another example of mutualism in nature is the symbiotic relationship of the enterobacteria Photorhabdus sp. with the nematodes Heterorhabditis. These bacteria live in the gut of the nematode. When the nematode bites insect larvae encountered in the soil, it regurgitates the entomopathogenic Photorhabdus inside the larvae, which kill and convert the larvae into nutrients for the worm (Ruby, 1996; Waterfield et al., 2009).
Host defense mechanisms are activated upon bacterial colonization but in the case of a mutualistic relationship, attenuation of the inflammatory process ensures tolerance (Chervonsky, 2012; Pedron
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and Sansonetti, 2008). For example tolerance in the intestine is achieved on the one hand by the fact that the microbiota can modify their extracellular polysaccharides in order to make them less immunogenic and to attenuate host proinflammatory signals, on the other hand by the fact that host epithelial cells produce a mucus preventing bacteria from engaging into direct contact with the cells.
This mucus also contains antimicrobial peptides that prevent overgrowth of bacteria (Lebeer et al., 2010).
Other bacteria from the environment (soil, water), which enter occasionally the gut, for example following ingestion of crude vegetables and fruits, are recognized by the immune system that triggers their elimination and prevents the development of a disease. For example, Pseudomonas aeruginosa can enter the gut and is then recognized and destroyed by host phagocytes. Host phagocytes in contact with bacteria like Pseudomonas but not commensals produce mature interleukine 1 (IL-1), a major cytokine triggering inflammation. Franchi and colleagues showed that detection of mature IL-1 can thus be used as a marker of non-commensal bacteria (Franchi et al., 2012).
Bacteria like Pseudomonas aeruginosa remain harmless as long as the host regulates their growth, but as soon as the defense of the host fails, for example in immunocompromised patients, bacteria invade and damage host tissues, leading to the development of a disease. These bacteria are then referred to as opportunistic pathogens. Pseudomonas aeruginosa is a common opportunistic pathogen in human, which is responsible for chronic infections in cystic fibrosis patients and is one of the most frequent agent leading to nosocomial diseases, infections acquired at the hospital where patients have often impaired immune defenses (Driscoll et al., 2007) .
I. A. 2. True pathogenic bacteria and emergent pathogens
Contrary to opportunistic pathogens, true pathogenic bacteria can evade the immune system and create a disease even in a healthy individual. For example, Yersinia pestis, the causative agent of bubonic plague transmitted by fleabites, was responsible for the death of millions of people in the middle age (Raoult et al., 2000). Today, despite the existence of antibiotherapy, pneumonia infections by Y. pestis are still life-threatening (Prentice and Rahalison, 2007). True pathogens can also target non-human hosts, for example Pseudomonas entomophila, which causes the rapid death of infected insects.
Another category of pathogenic bacteria includes emergent pathogens. When a bacteria, never described within the last 20 years, is found to be the causative agent of a disease in humans, it is named emergent pathogen (Lederberg, 1998). In addition to classical mechanisms driving genome evolution, the rapid evolution of pathogenicity in bacteria can be caused by horizontal transfer of
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genetic material that allows the appearance of new bacterial species (Bezuidt et al., 2011) (Wiedenbeck and Cohan, 2011). The genetic material transferred through the bacterial cell wall can correspond to plasmids or to phage DNA, originating from other bacteria. This process allows the production by the bacteria of new proteins, in particular new virulence factors. For example, genetic analysis of Yersinia pestis suggest that it was derived from Yersinia pseudotuberculosis, an enterobacteria, that lost genes involved in gut colonization while acquiring plasmids coding for virulence factors allowing lung colonization and development of plague (Prentice and Rahalison, 2007).
One of the emergent human pathogen of these last 23 years is the gram-negative Photorhabdus asymbiotica, responsible for ulceration of the skin. There are three Photorhabdus species, all entomopathogenic and living in symbiosis within the gut of soil nematodes. Only Photorhabdus asymbiotica has been recovered from infected wounds in human patients (Gerrard et al., 2003).
Genetic analysis comparing the three Photorhabdus species revealed that Ph. asymbiotica bacteria carry at least ten genes, not present in the two other Photorhabdus strains, coding for virulence factors found in other pathogenic bacteria for humans such as Pseudomonas aeruginosa and Yersinia pestis (Tounsi et al., 2006).
I. A. 3. Virulence factors of Pseudomonas sp. and Photorhabdus sp.
Bacteria have been divided in several groups based on different criteria such as their DNA composition, lipid composition and cell wall composition (Schleifer, 2009). One of the main classifications of bacteria is based on the presence of either a thick or a thin peptidoglycan layer.
Hans Christian Gram in 1884 elaborated a staining protocol to differentiate bacteria based on the thickness of the peptidoglycan. Bacteria with a thick peptidoglycan are stained at the end of the treatment and are named Gram-positive, while Gram-negative bacteria are bacteria with a thin peptidoglycan, which loose the staining during treatment. During my PhD I worked exclusively with Gram-negative Pseudomonas and Photorhabdus bacteria. More specifically I used two strains of Pseudomonas: P. aeruginosa and P. entomophila, and one strain of Photorhabdus: Ph.
asymbiotica. I did not study specifically the virulence factors of these bacteria, and their role in pathogenesis, rather I focused on developing a method to measure the overall virulence of these bacteria.
Virulence factors include all molecules produced by bacteria that promote bacterial multiplication within the host, and interfere with the host physiology. Virulence of bacteria is achieved by adhesion, release of toxic compounds, invasion of host tissues, and perturbation of the immune
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system. Virulence factors of Pseudomonas aeruginosa have been studied extensively, while much less is known on the virulence of Pseudomonas entomophila, isolated and identified in 2005 from fruit flies and decaying fruits in Guadeloupe (Vodovar et al., 2005). Photorhabdus asymbiotica has been first described as pathogenic for insects. Between 1977 and 2003, twelve cases of human infections with Ph. asymbiotica have been reported in Australia and in the United States (Gerrard et al., 2003). I am providing here a very brief review of virulence mechanisms focused mainly on P.
aeruginosa and secondarily on these two other bacterial species.
a. Adhesion systems
Adhesion of bacteria to host cells is often a prerequisite for infection and can be mediated by several molecules. These molecules include flagella, pili, and lipopolysaccharides (Fig. 1). In addition, several secretion systems ensure binding of bacteria to the host cell, also allowing bacteria to inject toxic compounds directly in the cytoplasm of the cell. Finally, bacteria produce a specific extracellular matrix allowing them to stick to the host epithelium, which is named biofilm.
• Flagella and pili
Major adhesion molecules described in Pseudomonas aeruginosa are flagella and type IV pili (Bucior et al., 2012). Flagella are involved in the motility of the bacteria as well as in its adhesion to the host cell, via the interaction of flagellin, a major component of the flagella, with Muc1, a glycoprotein anchored in the plasma membrane of the host epithelial cell (Lillehoj et al., 2002).
Type IV pili bind N-glycans of host cell. They are composed of pilin proteins produced in the cytoplasm of bacteria, translocated and assembled at their outer membrane by the well-conserved usher-chaperone (Bucior et al., 2012; Finlay and Falkow, 1997; Kline et al., 2009). Genomic analysis of P. entomophila revealed also three genes encoding putative adhesion molecules (filamentous hemagglutinin-like adhesins) as well as two putative autotransporter proteins with pertactin-type adhesion domain, a molecule similar to an adhesion molecule found in the bacteria Bordetella pertussis, the causing agent of wooping cought.
• Lipopolysaccharides (LPS)
LPS are molecules anchored in the outer membrane of Gram-negative bacteria cell wall and composed of lipid A, anchoring the LPS in the outer membrane, a core oligosaccharide, which varies in length, and a distal O-antigen made of repeats of tetrasaccharides units (e.g. mannose, fucose). LPS exhibit the same overall structure in all bacterial species and are specifically recognized by receptors present at the plasma membrane of host-cell: CD14 and TLR4 (Lloyd and
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Kubes, 2006). In particular recognition of LPS of Pseudomonas aeruginosa by TLRs host cell is essential to trigger efficient inflammation response (McIsaac et al., 2012)
No information is available on the putative role of LPS in adhesion process of P. entomophila as well as Ph. asymbiotica to host cell.
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• Type III and VI secretion systems (T3SS and T6SS)
Among the toxins secreted by bacteria, some require to be directly injected into the host cell to exert their pathogenic activities. The direct injection into host cell can be mediated by secretion systems; three have been described to date, the type III, IV and VI secretion systems (T3SS, T4SS and T6SS). Pseudomonas aeruginosa express the T3SS and the T6SS. The structure and function of the T3SS have been extensively studied in numerous bacterial species like Yersinia pestis, Pseudomonas aeruginosa, Salmonella enterica and Escherichia coli (Izore et al., 2011). The T3SS, or injectisome, is made of over twenty proteins that form a basal body, an export apparatus and a translocon. The basal body assembles first, it is composed of two rings inserted in the two membranes of the bacteria, then the export apparatus piles up, which forms the needle structure and finally, the translocon-forming protein inserts into the membrane of the host cell (Izore et al., 2011).
Several effector proteins transported through the T3SS of P. aeruginosa have been identified and will be described in the part c. Genetic analysis of Ph. asymbiotica revealed the expression of an orthologue of the protein Escv, a component of the T3SS found in the enteropathogenic E. coli.
Moreover, a protein hortologue of SopB has been identified in Ph. asymbiotica. SopB is an effector protein of the T3SS of Salmonella enterica. All together these results suggest the expression of a T3SS in Ph. asymbiotica (Tounsi et al., 2006). On the contrary, no T3SS loci have been detected in P. entomophila (Vodovar et al., 2006).
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Figure 1: Bacterial adhesion molecules. This scheme represents the different molecules expressed by Gram-negative bacteria that allow their adhesion to host cells. Bacterial flagellum as well as LPS promote adhesion by binding to host cell receptors such as Toll-like receptors (see part chapter II B.
a). Type IV pili mediates specific adhesion by binding to carbohydrates present at the host cell surface. Finally, exopolysacharides produced by bacteria are major components of biofilms supporting bacterial attachment to host cell.
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Much less informations are available on the T6SS, which has been recently discovered in a Vibrio cholera strain whose cytotoxicity required direct contact with host cell (Pukatzki et al., 2006).
The molecular structure of the T6SS, as well as its mechanism of assembly are unknown. However several studies suggest that the hemolysin coregulated protein (Hcp) and the valine-glycine rich protein G (VgrG) are structural secreted components of the T6SS (Pukatzki et al., 2009). Hcp molecule forms an hexameric ring as revealed by X ray crystallography that may compose the tubular structure of the secretion system. In the proposed model for T6SS assembly, Hcp proteins would assembly beneath VgrG proteins complex, whose structure is similar to the needle-like complex of T4 bacteriophage involved in puncturing membranes. Once the T6SS has crossed the host cell membrane the VgrG proteins may detach and exert toxic effects by crosslinking actin as demonstrated with the VgrG expressed by Vibrio cholera. T6SS loci have been detected in P.
aeruginosa as well as in P. entomophila (Mougous et al., 2006; Sarris and Scoulica, 2011). The effector proteins transported through these T6SS remain to be elucidated. No T6SS loci have been identified in Ph. asymbiotica to date.
• Biofilms
Another system of attachment of bacteria to cells is the production of biofilms that consist of a matrix of extracellular polymeric substances (EPS) made of polysaccharides, DNA and proteins.
Bacteria embedded in EPS are protected from environmental stresses such as antibiotics and from the host immune response (de Kievit, 2009; Ghafoor et al., 2011). Analysis of the relative contribution of these three polysaccharides in biofilm formation revealed that Psl is essential for initiating adhesion to the host cell. Adhesion seems likely provided by adsorption of Psl to bacteria as well as to host cell surface like lung epithelium or medical devices like catheters. Following attachment, bacteria multiply and synthesize a biofilm. Bacteria can then evade from the mature biofilm, return to a planktonic stage, and colonize a new site (Sauer et al., 2004).
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b. Two component systems and quorum sensing
The expression of virulence factors is tightly controlled by complex regulating systems, in particular by the two component systems (TCS) and the quorum sensing (QS) systems (Fig. 2).
• Two components systems (TCS)
TCS are composed of two proteins: a membrane sensor kinase and a cytoplasmic response regulator.
Upon extracellular stimulation, the sensor kinase autophosphorylates its cytosolic C-terminus, and the phosphate is then transferred to the N-terminus of the regulator, which acquires the ability to bind specific DNA promoter regions and activates genes transcription (Stock et al., 2000). In P.
aeruginosa 64 sensor kinases and 72 response regulators have been identified (Gooderham and Hancock, 2009). The Global activator S-Global activator A (GacS-GacA) two components system has extensively been studied and is composed of the sensor kinase GacS and the cytosolic regulator GacA (Heeb and Haas, 2001). This system was first identified in the plant pathogen Pseudomonas syringae where it participates in bacterial virulence by secreting enzymes damaging the host. The same system has been identified in P. aeruginosa, where it is involved in exotoxin production as well as in positively regulating the production of autoinducers, components of the quorum sensing discussed below (Heeb and Haas, 2001; Reimmann et al., 1997).
The GacA-GacS system has also been identified in P. entomophila. Lack of GacA in P.
entomophila abolishes its pathogenicity towards Drosophila (Vodovar et al., 2005).
A two component system has been identified in Ph. asymbiotica, KdpD/KdpE, which is involved in potassium sensing and evasion of bacteria from phagocytic destruction (Vlisidou et al., 2010).
• Quorum sensing
Quorum Sensing (QS) allow communication between bacteria by controlling the expression of an array of genes in response to bacterial cell density. These systems have been first identified in the bacteria Vibrio fisheri, where bacterial density is responsible for the control of bioluminescence (Finlay and Falkow, 1997). The QS molecular is conserved among bacterial species and is based on two main actors: an autoinducer (I), which is responsible for the production of an acylated homoserine lactone (HSL), which diffuses trough membranes, and a regulator (R), which acts as a receptor for the HSL. The HSL-regulator complex has the capacity to bind to specific DNA regions, and to induce the expression of genes encoding virulence factors. In Pseudomonas aeruginosa three QS systems have been identified, LasR- LasI, RhlR-RhlI and PqsE-PqsR. They elicit the production of exotoxins and are involved in biofilm formation (Finlay and Falkow, 1997; Miller and Bassler, 2001). Quorum sensing machineries have been identified in Photorhabdus species but not in Ph.
asymbiotica, nor in P. entomophila (Krin et al., 2006).
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Figure 2: Regulatory systems controlling expression of virulence factors in P. aeruginosa.
Expression of genes encoding virulence factors is under the control of finely tuned and interrelated systems: two-component systems (TCS) and quorum sensing (QS). This scheme depicts TCS and QS regulation in P. aeruginosa.
A TCS is composed of a membrane sensor kinase (e.g. GacS), which auto-phosphorylates when activated by external stimuli. The phosphate is then transferred to a cytoplasmic response regulator (e.g. GacA). In P. aeruginosa, the phosphorylated GacA has been shown to trigger the expression of a wide array of genes including genes of the QS.
The QS is a cell-to-cell communication system. It includes soluble homoserine lactone molecules (HSL) that freely diffuse across bacterial membranes and bind to regulators such as LasR, RhlR.or PqsR. This complex then promotes the transcription of virulence genes.
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c. Exotoxins
Exotoxins are molecules toxic for the host cells, secreted by bacteria in the extracellular medium or directly inside the host cell. Exotoxins are very diverse: proteases, glycolipids, siderophores.
Secretion machineries that cross the inner and outer membranes of bacteria allow the export of exotoxins outside the bacteria. Different secretion systems exist such as the type I or II secretion systems allowing the export of molecules in the extracellular medium, or the T3SS and the T6SS allowing the secretion of bacterial molecules inside the host (described in A. 3. a and fig. 1) (Bleves et al., 2010).
In Pseudomonas aeruginosa, the LasI-LasR system promotes the transcription of several genes including the LasB and the AprA genes. LasB encodes an elastase secreted via the T2SS, which has been shown to degrade surfactant proteins in the lung, preventing opsonization of the bacteria (Kuang et al., 2011) and AprA encodes an alkaline metalloprotease involved in the degradation of antimicrobial peptide from the host cell and released via the T1SS (Miller and Bassler, 2001). The RhlI-RhlR system promotes the transcription of several genes including the rhlAB gene involved in the production of rhamnolipids (Miller and Bassler, 2001). Rhamnolipids are glycolipids that act as biosurfactant, first identified in P. aeruginosa, they have been shown to participate in bacterial motility but also in the invasion of lung epithelium, making them highly toxic for host cells (Abdel- Mawgoud et al., 2010).
Also in P. aeruginosa, the well-studied T3SS mediates the transport of four effector proteins identified to date ExoS, ExoT, ExoY and ExoU. (Lee et al., 2005). ExoS and ExoT are enzymes acting on small G proteins and thus altering cytoskeleton and signaling pathway of host cell (Fraylick et al., 2002; Kazmierczak and Engel, 2002), ExoY is an adenylate cyclase that triggers endothelial permeability leading to edema (Ochoa et al., 2012) and ExoU a phospholipase that interferes with inflammatory response (de Lima et al., 2012).
A number of genes coding for putative exotoxins have been identified in P. entomophila by analyzing its genome, such as insecticidal toxins, alkaline proteases (homologues of the AprA metalloprotease in P. aeruginosa), or hemolysins (Vodovar et al., 2006). Some of them have been studied more extensively and their roles in the pathogenicity of P. entomophila towards Drosophila have been demonstrated, such as the role of hydrogen cyanide (Ryall et al., 2009), the entolysin hemolysin (Vallet-Gely et al., 2010) and the monalysin pore-forming protein (Opota et al., 2011).
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The exotoxins produced by Ph. asymbiotica are much less documented than those of Pseudomonads, especially those required for human infection. The analysis of their ability to disable or kill macrophages or hemocytes, the professional phagocytic cells of invertebrates, allowed the identification of putative virulence factors. Costa and colleagues showed the ability of Ph. asymbiotica but not Ph. luminescens, the restricted entomopathogen, to invade macrophages and hemocytes, replicate intracellularly and induce apoptosis of the host cells (Costa et al., 2009).
They suggest that the proapoptotic activity could due to exotoxins secretion including the homologue of the phospholipase ExoU. Ph. asymbiotica otherwise secrete same exotoxins than other Photorhabdus species required for insects killing. Among these exotoxins there are the gut- active toxin complex A (Tca), the multidomain Mcf1 and a metalloprotease PrtA involved in the gut destruction of insects, a hallmark of Photorhabdus infections (Waterfield et al., 2009).
Many other exotoxins are produced by bacteria, all of them aiming to damage the host or manipulate the host system. Since 2004, a virulence factor database (VFDBs) has been created (Chen et al., 2012), gathering all information on virulence factors of major pathogenic bacteria.
I. B. Measuring bacterial virulence
I. B. 1. Animal models to study bacterial virulence
The identification of genes involved in bacterial pathogenicity is essential to decipher the infectious processes they generate and to identify new therapeutic targets. Mice, rats and to a lesser extent primates are the main mammalian animal models used to study the etiology of human diseases, based on the notion that their close phylogenetic proximity to human makes them particularly relevant. For practical as well as ethical reasons, non-mamalian models to study human diseases have also been developed and used extensively. Among these models, the most commonly used are the vertebrate zebrafish Danio rerio, the fly Drosophila melanogaster, the worm Caenorhabditis elegans, the unicellular amoeba Dictyostelium discoideum and the plant Arabidopsis thaliana (Fig.
3). It has been shown that environmental opportunistic pathogens like Pseudomonas aeruginosa can infect all these organisms, and that they largely make use of the same virulence factors in these different organisms (Hilbi et al., 2007). In addition, in these models, simple assays have been developed to identify bacterial virulence genes by screening random bacterial mutants. The same tools can be used to search for new antimicrobial compounds inhibiting bacterial virulence, and to identify host genes involved in the defense against pathogens (Kurz and Ewbank, 2007). In this
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section I describe the main animal models and highlight their advantages and disadvantages as model systems to evaluate bacterial virulence. This is also summarized in the table 1.
a. Mus musculus
Rodents (mostly mice and rats) are the most common mammalian model organisms used in biomedical research. Mice are small mammals; gestation is completed within 20 days, a litter comprises 5 to 12 pups, and it takes 45 days for the pups to become adults. The complete sequence of the mouse genome was obtained in 2002 and it is very similar to that of the human genome: 75%
of the genes found in mouse have orthologs in human (Marshall, 2002; Waterston et al., 2002).
Diverse techniques have been developed to identify new genes and their functions, such as specific mutagenesis techniques (knock-out, knock-in, random mutagenesis), or RNA interference (Buer and Balling, 2003; Stein et al., 2003; van der Weyden et al., 2002). Because of anatomic resemblances, and due to the similarities between the murine and human immune systems (both innate and adaptive), mouse is a well-used model to study human infectious diseases. Specific mouse models have been created to mimic human infections and assess the virulence of pathogenic bacteria such as Pseudomonas sp. For example the virulence of P. aeruginosa has been assessed in a burned mouse model (Neely et al., 1999; Stieritz and Holder, 1975), in a mouse model of acute pneumonia (Comolli et al., 1999; Smith et al., 2004) and in mouse cornea infections (Gupta et al., 1996).
Infections are usually initiated by injecting bacteria, by feeding them to mice, or by allowing mice to inhale them. Survival of mice is then recorded over the days following the infection, as well as various physiological parameters (body temperature, weight, behavior).
Today, utilization of mammalian models is mandatory in some situations, such as in preclinical trials. In basic research, for practical and ethical reasons, the 3R strategy (“Replace, Reduce, Refine”) recommends the use of alternative non-mamalian animal models when possible.
Furthermore, the use of mammalian models is expensive and requires specific installations and organizations. Compared to other systems, mammals are difficult to manipulate and genetic manipulations are long and expensive (Waltz, 2005).
b. Danio rerio
The zebrafish Danio rerio is a small fish (4 to 6 cm), living in fresh water and feeding upon zooplanktonic crustaceans. It reproduces every two-three months, and a hundred of eggs are released by the hermaphrodite every 2 or 3 days. Embryos are transparent and develop externally,
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allowing the direct visualization of organs development, making zebrafish a tractable model to study organogenesis in vertebrates. Zebrafish is also used to study human diseases such as cancer and metastasis, wound healing or bacterial infection (Goldsmith and Jobin, 2012). Finally, zebrafish has both adaptive and innate immunity both largely similar to the mammalian immune system (Pradel and Ewbank, 2004).
Genetic informations on the zebrafish are available on line: the zebrafish model organism database (http://zfin.org). Zebrafish can be mutated using mutagenic chemical compounds. Alternatively, specific genes can be silenced by using RNAi or morpholinos. The material required for zebrafish aquaculture is not expensive: about 1000 USD for a 80 Zebrafish tank (Kim et al., 2009).
Zebrafish has been used to study virulence of pathogenic bacteria. The most studied is Mycobacterium marinum, which is closely related to the human pathogen Mycobacterium tuberculosis, the etiologic agent of tuberculosis. M. marinum generates tuberculosis-like infections in fish (Meijer and Spaink, 2011; Rodriguez et al., 2008). Zebrafishes are infected by injecting bacteria intraperitoneally and their survival is recorded. Bacteria expressing fluorescent proteins (e.g. GFP) allow the direct visualization of bacterial invasion of zebrafish embryos or of Casper zebrafishes. Virulence of other pathogens, like P. aeruginosa, has also been assessed in zebrafish models (Brannon et al., 2009; Clatworthy et al., 2009).
c. Drosophila melanogaster
Drosophila has been a model system for studying genetics and development since the end of the 19th century. Thomas Hunt Morgan received the Nobel price of Physiology or Medicine in 1933, for his work on heredity using Drosophila as a model organism (Morgan, 1910).
Drosophila are small flies (3-4 mm), have a short life cycle (2 weeks), and produce numerous eggs (a female can produce 500 eggs in 10 days). The genome of Drosophila has been sequenced in 2000 (Adams et al., 2000). It is composed of 120 Mb, distributed on 4 pairs of chromosomes (3 autosomal and one sexual), encoding about 13’600 genes. Informations on Drosophila are gathered online at ww.flybase.org. Most of mutants have been obtained by insertion of transposable elements but more recent methods have developed such as the use transgenic RNA interference (RNAi) (Dietzl et al., 2007; Ryder and Russell, 2003). A large stock of mutants is also available (flystock).
Drosophila is also used as a model system to decipher host-pathogen interactions (Bier and Guichard, 2012). Drosophila possess only an innate immune system, which involves two main pathways: the Toll-pathway (Lau et al., 2003), and the Immune deficiency-dependent (Imd) pathway (Hoffmann, 2003).
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Figure 3: Phylogenetic tree indicating major model systems.
This phylogenetic tree indicates the relative proximity between eukaryotic organisms. Most eukaryotic organisms belong to the four delimitated kingdoms: plant, amoebozoa, fungi and animals. Dictyostelium diverged from the line leading to animals after plants and before fungi. In the animal kingdom, four phyla are shown. In blue the chordata (e. g. human, mouse or fish), in pink the urochordata (e. g. ciona), in green the arthropoda (e. g. mosquitos) and in yellow the nematodes (e. g. Caenorhabditis elegans). Daphnia, not represented on this tree, belongs to the arthropod phylum. (Eichinger et al. 2005)
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Toll receptors have been first identified in Drosophila were they have been found associated to the dorsal-ventral polarity during Drosophila development (Anderson et al., 1985). In 1996 Jules A.
Hoffman and colleagues showed that Toll-receptors are essential for pathogen recognition by Drosophila cells, as well as for triggering signaling pathways leading to activation of the immune response. The predominant role of Toll and Toll-like receptors in pathogen recognition has then been identified in other organisms (Hansson and Edfeldt, 2005). Drosophila has been used to study not only the innate immune response during bacterial infection (Kounatidis and Ligoxygakis, 2012), but also the virulence factors of bacteria like P. aeruginosa (Kim et al., 2008), P. entomophila (Opota et al., 2011) and Ph. asymbiotica (Vlisidou et al., 2012). Two main methods are used to infect flies: feeding them with contaminated food or pricking their thorax with a contaminated needle. Survival of flies is recorded then (as a function of time).
d. Caenorhabditis elegans
The worm Caenorhabditis elegans is a small (1 mm) transparent nematode, which feeds upon bacteria and is easily maintained in petri dishes. It is mainly a hermaphrodite organism which produces about 300 self-fertilized eggs every 3 days at 25°C (Pradel and Ewbank, 2004; Riddle, 1978). Its genome has been fully sequenced and annotated in 1998 (consortium, 1998) and a large body of information on this model is available on a specific website (http://www.wormbase.org/).
Sydney Brenner introduced this organism as a model system at the beginning of the 70’s (Brenner, 1974) and received 30 years later the Nobel price for the advances made in understanding organ development and programmed cell death.
One of the remarkable features of this organism, making it a very tractable tool for genetic analysis, is the targeted gene silencing provided by feeding it with bacteria expressing RNAis (Fortunato and Fraser, 2005). This method has actually been originally developed in the worm C. elegans (Fire et al., 1998). Like Drosophila, C. elegans possesses only an innate immune system and this has allowed the analysis of the precise contribution of this system in the response against pathogens (Pukkila-Worley and Ausubel, 2012).
Virulence assays are performed with C. elegans by feeding them with the bacteria of interest and candidate antimicrobial drugs, then automated systems can record survival of nematodes (Ewbank and Zugasti, 2011; Garvis et al., 2009; Moy et al., 2009). Analysis of virulence of P. aeruginosa in C. elegans revealed that this bacteria uses similar virulence mechanisms against mammalian host, plants and nematodes. For example Exotoxin A of P. aeruginosa has been shown to be required for virulence of the bacteria in all three models suggesting that these distant organisms have conserved the molecular receptors for the toxin (Tan et al., 1999a; Tan et al., 1999b).
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e. Arabidopsis thaliana
Arabidopsis thaliana is a widely used plant model in laboratories. Its genome is small ("157 Mb) compared to other plants genomes (Bennett et al., 2003). Numerous mutants have been created and are commercially available. Mutant lines are mostly insertion mutants obtained using the Agrobacterium-mediated transferred DNA (T-DNA) method, and they can be easily maintained by self-fertilization. When needed, genetic screens allow the identification of mutated genes of interest (Krysan et al., 1999; Page and Grossniklaus, 2002). About 74% of the 29 454 annotated genes of A.
thaliana were mapped in 2003 using the T DNA method (Alonso et al., 2003). Genetic informations are available on internet, about A. thaliana, the mutants and the methods (http://www.arabidopsis.org/).
A. thaliana is easy and cheap to cultivate: it is small, 1000 plants can be cultivated in one square meter. Its generation time is about 5 to 6 weeks and each plant produces up to 10’000 seeds. Finally, to study host-pathogen interactions, pathogens can be easily injected in the leaf or in the roots, and rotting of the plant at the site of infection is observed within days (2 to 5). Research on plant- pathogens interactions revealed clearly different pathogen recognition receptors (PRRs) in mammals and plants. However PRRs identified in mammals and plants exhibit similar mechanisms of activation and signal transduction (Monaghan and Zipfel, 2012; Ryan et al., 2007). The virulence of several human pathogens, like Staphyloccocus aureus (Prithiviraj et al., 2005), Pseudomonas aeruginosa (He et al., 2004; Hendrickson et al., 2001; Yorgey et al., 2001) and Enterococcus faecalis (Jha et al., 2005) can also be assessed in A. thaliana.
f. Dictyostelium discoideum
D. discoideum belongs to the amoeba family which are professional phagocytes. It feeds upon bacteria in the soil (Raper, 1935). Its genome has been fully sequenced in 2005 (Eichinger et al., 2005). It is a unicellular organism with an A/T rich haploid genome of 34 Mb, a hundred time smaller than the human genome, distributed on 6 chromosomes and encoding about 8000 to 10000 genes (Eichinger et al., 2005). Sequence analysis revealed a high similarity between the Dictyostelium genes and their orthologs in vertebrates, suggesting that Dictyostelium is more closely related to vertebrates and mammals than to plants (Baldauf and Doolittle, 1997; Raper, 1935).
During my PhD I used Dictyostelium as model of mammalian phagocytes. The Dictyostelium strain we commonly use in our laboratory is the DH1 strain, derived from an environmental strain and selected for its ability to grow in liquid medium, and further mutagenized to make it uracil-
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auxotroph. Dictyostelium cells divide approximately every 12 hours at 21-22°C (a temperature above 26°C is lethal). One of the main advantages of Dictyostelium as a model system is its haploid genome. In haploid organisms, mutations introduced experimentally in one allele cannot be masked by the second unmutated allele, and characterization of gene function is considerably easier (Eichinger et al., 2005; Taft et al., 2007).
One of the amazing particularities of Dictyostelium is its ability to form a multicellular organism, making it a model system for studying multicellular development (Fig. 4). Indeed, in some environmental conditions (starvation), about 100’000 cells can aggregate and form a slug. If starvation persists the slug evolves to form a fruiting body composed of a stalk (20’000 cells) with on the very end of it a head made of spores (80’000 cells). Cells composing the stalk are dead while cells in the head are dormant but alive (Chisholm and Firtel, 2004) (Fig. 4). Another field of investigations using Dictyostelium as a model system is the study of host-pathogen interactions.
Dictyostelium has first been described as professional phagocyte feeding upon bacteria in the soil (Raper and Smith, 1939), it is thus logical that this organism, acting as a predator for bacteria, has developed mechanisms to ensure bacterial killing and degradation.
Dictyostelium is now a widely used model system to study bacterial virulence. In the simplest settings, the virulence of bacteria can be assessed by spreading Dictyostelium cells on a lawn of bacteria (Froquet et al., 2009) (Fig. 5A). D. discoideum cells are allowed to grow and feed upon bacteria for several days. Permissive bacteria allow growth of D. discoideum which results in a phagocytic plaque in the bacterial lawn, corresponding to the area where D. discoideum ate bacteria (Fig. 5A). To perform accurate measure of bacterial virulence, different numbers of Dictyostelium cells are deposited (10000, 1000, 100 and 10 cells) in the middle of a lawn of bacteria. Depending on the virulence of the bacteria, Dictyostelium cells grow more or less, resulting in a more or less large plaque (Fig. 5B, white circles). With this test, five categories of bacteria can be distinguished, from non-virulent bacteria permissive for the growth of 10 Dictyostelium cells, to fully virulent bacteria that inhibit growth of even 10’000 Dictyostelium cells. A color scale from green to red indicates non-virulent bacteria to highly virulent bacteria respectively (Fig. 5B). Analysis of Pseudomonas aeruginosa mutants permissive for the growth of Dictyostelium discoideum revealed the importance of two virulence pathways: the quorum sensing notably the LasR gene, and the type III secretion system which translocates in the host cell the ExoU cytotoxin (Lima et al., 2011).
Indeed, bacteria lacking these molecules are no longer virulent for Dictyostelium. Interestingly, these mutants are also less virulent in mice.
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Figure 4: Dictyostelium: from unicellularity to multicellularity.
This figure illustrates the different life styles of Dictyostelium: vegetative (unicellular) or multicellular. Right panel: when nutrients are available, amoebae Dictyostelium discoideum grows as vegetative cells. In nature, amoebae feed upon bacteria. The left panel shows the different steps of multicellular development of starved cells, from the aggregation to the fruiting body, containing about 100’000 cells. Micrograph of developmental steps of Dictyostelium from M. Grimson, R.
Blanton, Biological Sciences Electron Microscopy Laboratory, Texas Tech University.
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Figure 5: Assessing bacterial virulence by using Dictyostelium
A) Dictyostelium cells are deposited in the middle of a bacterial lawn on agar medium. Bacteria permissive for the growth of Dictyostelium allow the formation of a phagocytic plaque (white hole).
B) By depositing different numbers of cells (from 10’000 to 10) a more accurate measure of the bacterial virulence is provided. A color scale, from green to red, reflects the virulence of the bacteria, from permissive to non-permissive respectively.
10’000 1000 100 10 Cells
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I. B. 2. Daphnia magna as a model system
One of the famous first reports on the use of Daphnia magna as a model system was due to Elie Metchnikoff, who isolated bacteria from Daphnia at the end of the 19th century (Metchnikoff, 1884).
Today, about 100 species of Daphnia have been identified, Daphnia magna is one of the most commonly used in laboratories. Daphnia are zooplanktonic crustaceans, belonging to the class of the branchiopoda and the subclass of phyllopodia, meaning that Daphnia exhibits flattened leaf-like legs. It belongs to the order of cladocera, meaning that its body is enclosed in an uncalcified shell or carapace, which is transparent and made of a chitin polysaccharide (Ebert, 2005). It lives in standing fresh water (lakes, ponds), where it feeds on algae and bacteria, and is preyed upon by fishes. Daphnia are small organisms ranging from about 1 to 5 mm. They possess a heart with an open blood circulation, the hemolymph. They also have one eye and a cerebral ganglion. A mouth, a filter apparatus and a gut compose their digestive tracts (Fig. 6). The legs of the Daphnia produce a water current allowing particles, like algae and bacteria, to be filtrated and to enter the gut via the mouth. Particles from 1 to 50 !m can be retained by the filtration system and reach the gut.
The lifespan of Daphnia is about two months. Daphnia magna has an asexual reproduction mode, which allows the production of a clonal population. One week after birth, the young Daphnia can produce its first brood, of about twenty youngs. Daphnia will give rise to new broods every two or three days, all along its life. The mode of reproduction mainly depends on the food availability. If conditions deteriorate Daphnia can switch to a sexual reproduction mode, which leads to the production of a diploid egg resistant to environmental stresses (Fig. 7). The genome of Daphnia pulex, another Daphnia commonly used in laboratory has been recently sequenced (Colbourne et al., 2011). This is the first crustacean genome sequenced to date.
To summarize, the asexual reproduction, the short life cycle, the rapid production of a large number of youngs, the transparent body, the simplicity and low cost of the growth medium as well as the nutrients, make Daphnia an interesting model system.
Daphnia has been used as a model system to study natural parasite coevolution, such as the bacterial parasite Pasteuria ramnosa (Ebert, 2008), and is also well used as a model in toxicology and in ecotoxicology, as an indicator of environmental changes (Martins et al., 2007). Daphnia magna has recently been added by the National Institute of health to the list of animal models for biomedical research (http://www.nih.gov/science/models/daphnia/).
As reviewed by Dieter Ebert (Ebert, 2011), despite the numerous reasons that make Daphnia an attractive model for laboratory studies, Daphnia as a model system suffered until recently, from the lack of genetics tools. Daphnia genetics are essentially based on the cross of clones but no reverse genetics exist. Electroporation of DNA in Daphnia has been performed in 2010 (Kato et al., 2010).
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In addition the recently published genome sequence of Daphnia pulex, the first crustacean genome (Colbourne et al., 2011) opens new perspectives for the use of Daphnia as a model system.
During my PhD I investigated the possible use of Daphnia as a new model system to study bacterial virulence (see Results I).
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Figure 6: Annotation of the Daphnia anatomy.
On this photography of Daphnia, some elements of its anatomy are indicated. Antenna are flattened leaf-like legs involved in Daphnia motility as well as in provoking a water current allowing particles to reach the filter apparatus via the mouth. The carapace is transparent allowing the visualization of organs, in particular the gut and the eggs in the brood chamber. (Ebert, 2005).
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Figure 7: Daphnia life cycle.
This scheme depicts the different life cycles of Daphnia. When food is available Daphnia undergo asexual reproduction mode (parthenogenesis). They produce about 20 clonal daughters every 2 or 3 days that develop in the brood chamber. Daughters become sexually mature after 2 weeks and in turn produce parthenogenetic daughters. In case of environmental stress conditions Daphnia female produce parthenogenetic son as well as haploid egg. Males fertilize haploid eggs resulting in diploid eggs that are laid on the ground of the pound. These are resting eggs. They stop their development (diapause) until environmental conditions become favorable again. From Dieter Ebert (2005) Ecology, epidemiology, and evolution of parasitism in Daphnia.
