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,
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,