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GENERAL DISCUSSION
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GENERAL DISCUSSION
We are constantly interacting with a wide variety of microorganisms including putative pathogenic bacteria (Introduction I). The outcome of these interactions depends on both the host organism and the bacteria, which is nicely summarized by the expression “the battle of the two genomes”.
Bacteria and their arsenal of virulence factors are facing host defense systems, and the result of this confrontation determines the outcome of infections (Introduction II). To envisage the development of new treatments it is crucial to decipher both the mechanisms of virulence of bacteria as well as the mechanisms of host defense.
During my PhD I had the opportunity to investigate both facets. First I investigated the possible use of Daphnia magna as model system to evaluate bacterial virulence (Results I). Then I studied several aspects of the host response using the amoeba Dictyostelium discoideum as a model (Results II, III and IV).
Daphnia as an alternative model to study bacterial virulence.
The ability of bacteria to inflict damage to a host is attributed to the expression of virulence factors that include a wide variety of molecules, such as exotoxins, finely regulated by complex systems such as the quorum sensing, a cell-to-cell communication system, or the two component systems, and that can evolve as a consequence of horizontal gene transfer (Introduction I. A.). Virulence factors enable pathogenic bacteria to escape bactericidal strategies of the immune system. The identified bacterial genes and mechanisms involved in bacterial virulence is essential to develop new therapeutic approaches and to elaborate new antibacterial treatments. Ideally, a drug could target specifically a mechanism essential for bacterial virulence, while avoiding deleterious effects on the host and on its normal bacterial flora.
The first step in elucidating virulence factors is thus to identify virulence factors. For this one must be able to assess and compare the virulence of numerous bacterial strains and mutants. To this aim virulence assays are performed by infecting animals with the putative pathogenic bacterial strains and recording the survival of the host. This first step requires numerous animals. Mice and other rodents are the mammalian host models most often used in laboratories, but their utilization is expensive and requires specific expertise and installations. Analysis of bacterial virulence in other model systems such as the fly Drosophila melanogaster, the worm Caenorhabditis elegans, or the zebrafish Danio rerio revealed that bacteria use largely the same virulence mechanisms to infect various hosts, from plants to human (introduction I. B). In addition, non-mammalian organisms are low costs compared to mammals and genetics can be more easily performed (Table 1).
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Water flea Daphnia magna are zooplanktonic organisms that have previously been used as model systems to study host-parasite co-evolution. Daphnia are very cheap and easy to cultivate, they produce about 20 individuals every 2 or 3 days. Surprisingly, Daphnia had never been used to study the virulence of opportunistic bacterial pathogens like Pseudomonas aeruginosa, although they would be expected to encounter these pathogens in their natural habitat.
There are practical advantages in using Daphnia to study bacterial virulence compared to other model organisms. Clonal populations of Daphnia magna are easy to cultivate since they engage into asexual reproduction and since each adult can produce about 20 Daphnia every 3 days. I used up to 100 Daphnia per experiment but depending on the culture size much larger could easily be obtained.
Culture media as well as food (algae) have a low cost. Finally, upon exposure to pathogens survival curves can be obtained in a one-day experiment, while it usually takes several days in other model systems. In that perspective, use of Daphnia may facilitates research on bacterial virulence.
During my PhD, I developed in our laboratory a method to assess bacterial virulence using Daphnia magna (Results I). I first set up in the laboratory the material required for the culture of Daphnia. I then developed an assay to evaluate bacterial virulence. To this aim I used previously identified pathogens for mice, Drosophila or Dictyostelium. I incubated Daphnia with these bacteria in Eppendorf tubes (3 Daphnia in 1 ml of medium) at specific bacterial concentrations. I showed that virulent opportunistic human pathogens such as Pseudomonas aeruginosa, as well as entomopathogenic bacteria, like Pseudomonas entomophila or Photorhabdus asymbiotica, are also virulent for Daphnia. In addition I showed that isogenic bacterial mutants that had previously been shown to be non-virulent for mice, Drosophila and Dicytostelium did not kill Daphnia (summarized in figure 33). My observations, coupled to the ease of use of Daphnia, and to the development of Daphnia genetic tools, indicate that this Daphnia can be used as a model to understand the complex interactions between pathogenic bacteria and infected hosts. My results also reinforce previous observations suggesting that opportunistic pathogens use similar virulence mechanisms to infect a variety of hosts.
Besides providing a fast and simple method to measure bacterial virulence, Daphnia can also be suited to study host defense mechanisms. Indeed, further analysis, using notably different Daphnia isolates exhibiting different sensitivities toward a same pathogen, could provide a better understanding of the host response. In deed, sensitive clones of Daphnia towards natural parasites have been identified (Luijckx et al., 2012). We are currently testing the sensitivity of different Daphnia magna clones to different opportunistic pathogens. Preliminary results suggest that different clones of D. magna may exhibit differential sensitivities to certain pathogenic bacteria.
If these results are confirmed, this line of research could in principle allow us to identify Daphnia genes involved in the defense against specific pathogens.
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Daphnia may also be used in screens for new antimicrobial drugs. Indeed, in our laboratory, an ongoing project is aimed at identifying new antibacterial drugs. The first screening is based on a Dictyostelium-based assay, to identify compounds decreasing the virulence of pathogenic bacteria.
To confirm and refine the results of this initial screening, Daphnia may provide a powerful tool for secondary screening.
Figure 33: Bacterial virulence in different model systems.
This table summarizes the virulence of several bacteria (Photorhabdus and Pseudomonas sp.) towards different hosts: mouse, Drosophila, Dictyostelium and Daphnia. Green squares indicate a bacteria non virulent in the model system used. On the contrary, red squares indicate bacteria that kill the infected host. Pathogenic bacteria can infect a variety of hosts and attenuated mutants lose concomitantly their virulence against all hosts. This indicates that results obtained in a Daphnia infection model can tentatively be extrapolated to other systems.
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Host defense
During my PhD I studied a second aspect of host-pathogen interactions, the response of the host to bacterial pathogens. In mammalian hosts, invasion by pathogenic bacteria activates the two main branches of the immune system: innate and adaptive immunity. Actors of the innate immunity (e. g.
professional phagocytes) build up the first line of defense, which is fast, but exhibits a low level of specificity. Adaptive immunity allows a slower but highly specific response, and the establishment of an immune memory. Both innate and adaptive immunities act in concert to ensure the complete elimination of the pathogen (Introduction II. A.).
During the early steps of infection, professional phagocytic cells like macrophages and neutrophils migrate to the site of infection where they recognize and ingest the microorganisms to be eliminated.
This process is named phagocytosis. Phagocytosis is an actin-based process that leads to the internalization of particles like bacteria in an intracellular membrane–bound organelle named the phagosome. After actin depolymerization, the phagosome matures by acquiring new components from its fusion with endocytic compartments (Introduction II. C.). This maturation process is essential to ensure intracellular bacterial killing (Introduction II. D.). In our laboratory we have been using the amoebae Dictyostelium discoideum as a model phagocytic cell for the last decade.
This second section is denser than the first one because it regroups three independent studies. First we dissected the relative contributions of the three proteins (Phg1A, SadA and SibA) in cellular adhesion to various particles. The results obtained led to a second project focused on the role of Phg1 in intracellular transport and sorting of membrane proteins like SibA. Finally, during my PhD I also clarified the specific roles of Phg1, Kil1 and Kil2 in elimination of bacteria Klebsiella aerogenes in the phagolysosome.
Phg1A and SadA control the expression of the SibA adhesion molecule.
Phagocytosis starts with the adhesion of the phagocyte to bacteria. This adhesion is achieved through the binding of phagocyte surface receptors to ligands at the surface of bacteria. In mammalian professional phagocytes, several types of receptors have been identified such as the Toll-like receptors, the scavenger receptors, the Fc$ receptors and the complement receptor 3 (C3R) (Introduction II. B. 1.). These receptors, in addition to providing adhesion to bacteria also generate intracellular signals, ultimately leading to the reorganization of the actin cytoskeleton, a hallmark of phagocytosis initiation. As a professional phagocyte, which engulfs bacteria in a very efficient way, Dictyostelium discoideum is an interesting model to study phagocytosis and more particularly to identify the adhesion molecules promoting internalization of particles.
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Few putative receptors have been identified in Dictyostelium, notably the PHG1A, SADA and SIBA gene products (Introduction II. B. 2). Indeed the corresponding knock-out strains exhibited similar cellular adhesion and phagocytosis defects. However the phagocytosis defects of these mutants does not necessarily imply a direct role of Phg1A, SibA and SadA proteins as surface receptors.
Our results (Results II) revealed that the surface expression of SibA was markedly diminished in phg1A and sadA KO cells. Further analysis revealed that the stability of the protein SibA was affected in these mutant cells and that this may account for the loss of SibA at the cell surface. This strongly suggest that Phg1A and SadA are not directly involved in adhesion to particles but rather act as regulators of the expression of SibA, which is to date the best candidate for the role of phagocytosis receptor recognizing hydrophilic particles (Fig. 34).
By creating and analyzing a chimera made of the transmembrane and cytoplasmic domains of SibA and of the unrelated extracellular domain of csA, we showed that the transmembrane or the cytoplasmic domain of SibA are sorted in the cell in a Phg1-dependent manner. This interesting result provided us the opportunity to address the molecular mechanisms of membrane protein sorting in Dictyostelium (Results III).
Phg1A is directly involved in the sorting of the SibA membrane protein
Secreted proteins are synthesized in the endoplasmic reticulum and transit through the Golgi where they undergo maturation process such as glycosylation before reaching their final destination in which they will carry out their work. The transport of proteins occurs in vesicles that bud from one compartment and fuse with the next one.
Using a classical cut and paste approach we created chimeras to determine the motifs recognized by Phg1A in the transmembrane and cytosolic domains of SibA (Results III). The results revealed that Phg1A recognizes the transmembrane domain of SibA and promotes its localization at the plasma membrane. In addition we showed that Phg1A interacts with the transmembrane domain of SibA and that glycine motifs present in this domain are involved in the interaction. Thus our results revealed a new sorting mechanism based on the recognition of transmembrane domains by Phg1A.
Further experiments suggested that Phg1A would notably allow SibA to exit the endoplasmic reticulum.
We are currently studying the intracellular sorting of various chimeric proteins in order to determine which transmembrane motifs, other than glycine motif, are recognized by Phg1A.
Moreover, the phg1A KO cells exhibit numerous defects such as the secretion of its lysosomal enzymes in the medium or the killing defect of specific bacteria. The results suggesting a role for Phg1A as sorting receptor bring new perspectives to elucidate the role of Phg1A in these cellular functions.
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Figure 34: Model of SibA-mediated adhesion controlled by Phg1A or SadA. Phg1A and SadA have been shown to regulate the expression of SibA. More precisely, Phg1A has been shown to be involved in cell surface localization of SibA (1). SibA is directly involved in adhesion to substrate (2). Binding of SibA to its ligand triggers the recrutement and binding of Talin protein, which allows actin cytoskeleton reorganization, a process necessary for cell membrane extension notably during phagocytosis (3).
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Phg1A/B control the expression of Kil1, a sulfotransferase required for the killing of Klebsiella
After closure, the phagosome undergoes fusion and fission events with endocytic compartments.
After maturation of the phagosome, several mechanisms cooperate to ensure the destruction of ingested bacteria. Production of superoxide anions, acidification of the lumen, activities of hydrolytic enzymes are among the bactericidal mechanisms required for efficient bacterial elimination (Introduction II. D. 1). In Dictyostelium, maturation of the phagosome occurs through similar mechanisms than in mammalian phagocytes (Introduction II. D. 2). This is highlighted by the fact that numerous orthologs of mammalian bactericidal molecules have been identified in Dictyostelium. In addition, random mutagenesis allowed the identification of new genes involved in intracellular killing. Phg1, Kil1 and Kil2 are proteins that have been shown to exhibit a specific defect in killing of Klebsiella bacteria. The role of Phg1A in bacterial killing is unknown but, as mentioned above, phg1A KO cells exhibit intracellular transport defects that could account for their killing defects. Kil2 is a magnesium transporter in the phagosomal membrane required for Klebsiella elimination. Finally, Kil1 is a sulfotransferase that, when overexpressed in phg1A KO cells, restores the killing defect of the mutant.
During my PhD I investigated on the role of Phg1 and Kil1 in killing mechanisms of Klebsiella. I also investigated the putative interplay between Phg1 and Kil1 with Kil2 protein in this process. To this aim I analyzed several parameters involved in the lysosome physiology, such as the lysosomal enzymes content and activity, and the acidification of the endosomal compartments.
The results (Results IV) showed that the killing defect of phg1A KO is mainly due to the loss of Kl1 in this mutant cells. Consistent with this report, we showed that in the phg1A KO cells overexpressing Ph1B, which kill efficiently Klebsiella, the intracellular amount of Kil1 is increased.
In addition we showed that the loss of Kil1 cannot account for the other defects of phg1A KO.
Finally, we showed that Kil2 participates in Klebsiella elimination in a distinct mechanism than Phg1A, Phg1B and Kil1. The putative relation of these proteins with Klebsiella elimination is summarized on the last figure of the submitted publication.
Elucidating the role of Kil1 in bacterial killing is therefore the next important step. Sequence alignment revealed a high homology of Kil1 with the human N-deacetylase N-sulfotransferase 4 (NDST4) (Aikawa et al., 2001). This enzyme is involved in the replacement of N-acetyl glucose (GlcNac) with sulfate leading to heparan sulfate proteoglycans (HSPGs). Consistent with this proposal, no sulfated proteins were found in kil1 KO cells. Moreover, preliminary experiments of coimmunofluorescence tend to show a colocalization of these proteins with the Golgi but neither with the endoplasmic reticulum nor with endosomes p25 (Fig. 35 and 36).
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HSPGs are components of the membrane as well as the extracellular matrix and are involved in many physiological functions such as information transfer via integrin activation or metabolism regulation of food intake (Bishop et al., 2007). NDST-4 is to date poorly characterized, but structure and enzymatic activity comparisons revealed differences with the three other isoforms NDST1, NDST2, NDST3. NDSTs are ubiquituous in the organisms and the combination of NDST1/NDST2 is the most widely found. An exception is the mast cell, which is a component of the adaptive immune system involved in allergic reaction and which only expresses NDST2. Interestingly, mast cells lacking NDST2 proteins exhibit strong proteases storage defect in their granules (Henningsson et al., 2002). This effect could be explained by a role of HSPGs in the attachment of proteases to the luminal side of the granules, allowing their export from the Golgi. Thus we could hypothesize that the loss of Kil1 would lead to altered carbohydrates sulfation leading to abnormal lysosomal enzymes sorting, impeding bacterial killing. This hypothesis can be contradicted by the fact that the overexpression of Kil1 in phg1A KO did not prevent secretion of the lysosomal enzymes "-manosidase and #-glucosaminidase. This could be explained by a role of Kil1 in the transport of a specific unidentified lysosomal enzyme.
It has been shown that numerous lysosomal enzymes in Dictyostelium are sulfated (Freeze and Wolgast, 1986). Cardelli and colleagues showed that sulfated carbohydrates on lysosomal enzymes in Dictyostelium are not critical determinants for the transport and sorting of these proteins to lysosomes (Braulke et al., 1987; Cardelli et al., 1990). On the contrary they observed that the lack of sulfated residues impaired the delivery of the lysosomal enzymes in the lysosomes. Half of the enzymes are secreted compared to WT cells. There is no direct evidence to date to link lysosomal enzymes sulfation to their rate of secretion.
As mentioned by Cardelli et al, others proteins than lysosomal enzymes are sulfated in Dictyostelium, thus others unproperly sulfated proteins could be responsible for the observed phenotype of kil1 KO cells. For this reason, and because Kil1 is the only sulfotransferase in Dictyostelium, we suggest that a proteomic analysis of the main sulfated proteins in Dictyostelium as well as a proteomic analysis of proteins secreted by the kil1 KO, could bring new insights on the functions of sulfation in killing mechanisms in amoebae. We also propose to perform more genetics such as a suppressor screen, which could allow the identification of molecules involved in the same signaling pathway.
Together, the results obtained during my PhD showed that Phg1A participates in cellular adhesion by controlling cell surface expression of SibA. This is achieved by interacting with their TMDs and promoting their transport from the ER to the cell surface. Similarly Phg1A primarily plays a role in We suggest that Phg1A could act as a stabilizer of unconventional transmembrane domains,
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intracellular killing by determining the stability of the Kil1 protein, although we did not investigate further how Ph1gA controls the expression of Kil1. !
Figure 35: Kil1 localizes in intracellular compartments that are not recycling endosomes.
Immunofluorescence assay has been performed on the different strains in order to localize Kil1 in these cells. As control we used antibody against p25, a protein that has been shown to be associated to recycling endosomes (red). Antibody against Kil1 (white) reveals a similar localization of Kil1 in all cell types. Kil1 localizes to intracellular compartments that are not recycling endosomes.
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Figure 36: Kil1 colocalizes with Golgi marker but not endoplasmic reticulum marker. A) Immunolabelling using antibodies against Kil1 and protein disulfide isomerase (PDI) in WT and phg1A KO cells. B) immunofluorescence labeling in WT cells overexpressing Kil1 (WT + Kil1) with p25 marker (endosomes) and GFP-Golvesin (Golgi marker)
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notably those containing glycines motifs. We are currently trying to define an algorithm that could predict which TMDs are recognized by Phg1A.
During my PhD I showed that overexpression of Phg1B in phg1A KO restored partially the intracellular amount of Kil1. This confirmed previous reports suggesting synergistic roles of Phg1B and Phg1A.We showed that stability of SibA in phg1B KO cells was affected in a temperature-dependent manner. We are currently trying to determine the mechanisms by which Phg1B contributes to the sorting of SibA.
The role of Phg1 proteins was first identified in Dictyostelium. Orthologs are also found in other
The role of Phg1 proteins was first identified in Dictyostelium. Orthologs are also found in other