Listeria monocytogenes est une bactérie pathogène responsable de la listériose chez l’homme. Cette bactérie est capable d’envahir et de se répliquer dans des cellules phagocytaires et non-phagocytaires de l’hôte. De fait, Listeria interagit et interfère avec de nombreux composants cellulaires au cours de sa réplication. Mon projet de thèse s’est focalisé sur les altérations du protéome des cellules infectées par Listeria. Je me suis en particulier intéressé à l’activation de protéases de l’hôte et aux dégradations de certains facteurs cellulaires en réponse à l’infection.Une analyse protéomique de cellules humaines traitées avec la Listeriolysine O (LLO), une toxine secrétée par Listeria formant des pores dans les membranes cellulaires, nous a permis d’identifier plusieurs dizaines de facteurs cellulaires dégradés en réponse à l’exposition à cette toxine.Nous avons pu confirmer la dégradation de ces protéines dans le contexte d’une infection par Listeria in vitro (dans un modèle de cellules en culture) et in vivo (dans un modèle animal). Nous anticipons que la dégradation de ces protéines, impliquées dans différentes fonctions cellulaires, joue un rôle au cours de l’infection par Listeria en modifiant la physiologie des cellules hôte.Nous avons en parallèle mis en évidence que Listeria interfère avec le fonctionnement des lysosomes. Nous avons montré que la LLO déstabilise les lysosomes des cellules hôtes et induit leur perméabilisation. Ceci permet la libération de certaines protéases lysosomales, telles que les cathepsines, dans le cytosol des cellules hôte. Ces cathepsines, une fois libérées, pourraient alors cliver différentes protéines cytosoliques et ainsi modifier la physiologie des cellules infectées.L’ensemble de mes travaux a permis de montrer que Listeria interfère avec le fonctionnement de nombreuses protéases de l’hôte et induit la dégradation de plusieurs facteurs cellulaires. Ces cibles dégradées constituent une nouvelle classe de protéines dont nous pouvons maintenant tester le rôle potentiel dans l’infection. Mes travaux ont d’autre part permis de montrer que d’autres toxines bactériennes, similaires à la LLO, induisent également des altérations du protéome des cellules hôtes. Ceci suggère que les mécanismes identifiés avec Listeria sont conservés entre différentes classes de pathogènes bactériens. / Listeria monocytogenes is a foodborne pathogen responsible for human listeriosis which invade and replicate in both phagocytic and non-phagocytic cells. Listeria intracellular life cycle involves interference with host cell components. My PhD project aims to characterize host cell proteome modifications in cells infected by Listeria. I focused my research on host proteases activation and the degradation of host cell proteins in response to infection. A proteomic analysis performed on cells treated with purified Listeriolysin O (LLO), a pore forming toxin secreted by Listeria, identified more than 90 proteins degraded in response to the toxin. We validated the degradation of these proteins using both in vitro and in vivo models of Listeria infection. We anticipate that the degradation of these proteins can significantly impact the infection process through the modification of host cell physiology. In parallel, we identified that Listeria impairs lysosomal functions. We demonstrate that extracellular Listeria, via LLO secretion, alter lysosomal integrity in epithelial cells. LLO induces lysosomal membrane permeabilization and the release of lysosomal proteases in the host cytosol. The release of such proteases, which remain transiently active, may alter cellular physiology by degrading different cytosolic factors. Altogether, my results highlight how Listeria reshapes the host proteome by altering the activation or localization of host proteases and by inducing protein degradations. My data also establish that other bacterial toxins close to LLO also induce proteome modifications, thus unveiling that these mechanisms are shared among different class of bacterial pathogens.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

Delivery of macromolecules into the cytosolic space of eukaryotic cells is a pressing challenge in biopharmaceutics. Macromolecules are often encapsulated into liposomes for protection and improved distribution, but the their size often induces endocytosis of the vehicle at the target site, leading to degradation of the cargo. Listeriolysin O is a key virulence factor of Listeria monocytogenes that forms pores in the endosomal membrane, ultimately allowing the bacterium to escape into the cytosol. This function of LLO has been used to improve cytosolic delivery of liposomally encapsulated macromolecules in a number of instances, but its innate toxicity and immunogenicity have prevented it from achieving widespread acceptance. Through site-directed mutagenesis, this study establishes a mutant of LLO (C484S) with enhanced activity, allowing for a reduction in the amount of LLO used for future applications in liposomal drug delivery.

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Clostridium perfringens is the causative agent of gas gangrene. A lethal infection in mice requires a large inoculum suggesting that the immune system is involved in inhibiting disease. Human monocytic cells and neutrophils killed C. perfringens in vitro when complement was present. Macrophages and neutrophils co-localized with C. perfringens in vivo when bacterial numbers were low. Depletion of neutrophils and monocytes in mice revealed that monocytic cells play a role in inhibiting C. perfringens gas gangrene in mice infected with an intermediate dose. C. perfringens can persist in the tissues and this could be mediated by persistence within macrophages. To examine if the toxin perfringolysin O (PFO) could mediate this, less active variants of PFO were used to examine what occurs between phagosomal escape and cell lysis. The mutant forms of PFO did mediate phagosomal escape in macrophages and were found within macrophages at higher numbers than wild-type C. perfringens. Our data were preliminary but may indicate that less active PFO mediates intracellular persistence. To investigate the role of sialidase in C. perfringens gas gangrene we made nanI-, nanJ-, and nanI-/nanJ- mutants. We observed that NanI is responsible for the majority of sialidase activity of C. perfringens strain 13, that NanJ is an extracellular sialidase, and that these genes are transcriptionally regulated by sialic acid. Murine infection trials revealed that these sialidases may be protective for mice during infection. In conclusion, murine monocytes inhibit disease onset and C. perfringens sialidase enhances mouse survival. However, the toxin PFO if less active promotes the survival of C. perfringens with macrophages. / Master of Science

The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner. The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions. V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host. We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae. Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells.

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Die Entwicklung einer mukosal verabreichbaren, effektiven DNA-Vakzine gegen Infektionskrankheiten oder Tumorerkrankungen auf der Basis invasiver attenuierter Bakterien ist eine vielversprechende Alternative zu bisherigen parenteralen Strategien der genetischen Vakzinierung. Innerhalb dieser Arbeit wurden Salmonellen-Impfstämme für die orale Übertragung eines eukaryontischen Expressionsplasmids mit dem kleinen Oberflächenantigen des Hepatitis-B-Virus (HBsAg) als Modellantigen optimiert. Die kontinuierliche Sezernierung von Plasmiden als filamentöse Phagenpartikel wurde als ein erster Ansatz getestet, um mit lebenden Bakterien eine DNA-Vakzine innerhalb infizierter Zellen freizusetzen. Die Salmonellen-vermittelte Phagensekretion in der Wirtszelle ist jedoch nicht effizient genug, die Expression des Transgens zu vermitteln. Alternativ wurde ein Ansatz gewählt, durch eine spontan induzierte Lyse der Impfbakterien, Plasmid-DNA in die Wirtszelle zu übertragen. Dazu wurde ein neuartiges bakterielles Autolysesystem etabliert, basierend auf einem Zwei-Phasen-Expressionssystem und von Bakteriophagen abgeleiteten Lysedeterminanten. Dieses System ermöglicht erstmals die kontinuierliche Freisetzung von Plasmid-DNA und Proteinen aus einzelnen, lysierenden Salmonellen innerhalb einer sonst gesunden bakteriellen Gesamtpopulation. Innerhalb infizierter COS7-Zellen führt die Freisetzung des porenformierenden Proteins Listeriolysin O durch autolytische Salmonellen zur Zerstörung der Vakuole, in der die Impfbakterien replizieren, und erleichtert somit den Transfer der Plasmid-DNA aus den Bakterien in das Zytoplasma der Wirtszelle. Die Lysedeterminante und die eukaryontische Expressionskassette für HBsAg wurden auf einem Plasmid kombiniert, sowie eine Kassette zur konstitutiven Expression des Histon-ähnlichen Proteins aus Thermotoga maritima (TmHU) in ein solches Konstrukt integriert. TmHU stabilisiert die Plasmiderhaltung unter nicht selektiven Bedingungen und besitzt das Potential, die Effizienz der DNA-Translokation innerhalb der Wirtszelle zu erhöhen. Durch die orale Gabe optimierter autolytischer Impfbakterien konnte eine potente HBsAg-spezifische Antikörperantwort sowie eine zytotoxische zelluläre Antwort induziert werden. Bereits die einmalige Gabe der autolytischen Bakterien induzierte eine höhere antigenspezifische Antikörperantwort, als die herkömmliche intramuskuläre DNA-Vakzine. Das im Rahmen dieser Arbeit entwickelte Konzept autolytischer Salmonellen stellt also eine neuartige, effiziente Strategie für den mukosalen DNA-Transfer dar. Die Übertragung des Konzeptes der Autolyse auf andere bakterielle Trägersysteme ist möglich und kann zur Erweiterung des Anwendungspektrums bakterieller Vektoren beitragen. / The development of an effective mucosal DNA vaccine against infectious diseases or tumors based on invasive attenuated bacteria is a very promising alternative to common parenteral routes of genetic vaccination. This work aimed at the optimization of Salmonella vaccine strains for the oral delivery of an eukaryotic expression plasmid encoding the small Hepatitis B Virus surface antigen (HBsAg), here used as model antigen. The continuous secretion of plasmids as filamentous phage particles was first tested as a mean for the delivery of the DNA vaccine by living bacteria inside infected host cells. However, Salmonella-mediated phage secretion inside cells did not suffice for the induction of transgene expression. As alternative approach, inducible spontanous lysis of bacteria was used to mediate the release of plasmid DNA into host cells. For this purpose a novel bacterial autolytic system was established on the basis of a two-phase expression system and lysis determinants derived from bacteriophages. This system allows for the first time the continuous release of plasmid DNA and proteins from only few lysing Salmonella within an otherwise healthy bacterial population. Inside COS7 cells the release of the pore-forming protein listeriolysin O by autolytic Salmonella mediates the destruction of the Salmonella-harbouring vacuole, thereby facilitating the transfer of plasmid DNA from bacteria into the host cell cytoplasm. The lysis determinant was combined with the eukaryotic expression cassette for HBsAg on one plasmid. In addition, a cassette for the constitutive expression of TmHU, a histon-like protein derived from Thermotoga maritima, was integrated in such vector. TmHU stabilizes the plasmid propagation in the absence of selective pressure and has the potential to increase the efficiency of plasmid translocation inside the host cell. The oral administration of the optimized autolytic bacteria stimulated a potent HBsAg-specific antibody response as well as a cytotoxic cellular response. Already a single inoculation of the oral vaccine induced a higher specific antibody response than the conventional intramuscular DNA vaccine. Therefore the concept of autolytic Salmonella carrier strains developed in this work constitutes a novel efficient strategy for mucosal DNA delivery. The transfer of this concept to other bacterial carriers is possible and may widen the application field for bacterial vectors.

Endocytosis is a fundamental cellular process by which cells transport material from the outside to the inside of the cell through the formation of membrane invaginations that bud off from the plasma membrane. This process is important for nutrient uptake, regulating cell surface receptors and the overall plasma membrane composition. Cells have several different types of endocytic pathways where clathrin- mediated endocytosis is the most studied. Importantly, pathogens and secreted virulence factors bind to cell surface receptors and hijack the endocytic pathways in order to enter host cells. Depending on their size and molecular composition, pathogens and virulence factors are thought to make use of distinct endocytic pathways into the cell. This thesis focuses on early host cell interactions with virus, bacterial membrane vesicles and a pore-forming toxin, with a particular emphasis on endocytic mechanisms and plasma membrane repair. During entry of pathogens, it is thought that interactions with specific cell surface molecules drive the recruitment of endocytic proteins to the plasma membrane. Viruses possess a very defined molecular composition and architecture, which facilitate specificity to these interactions. We found that Adenovirus 37, a human ocular pathogen, binds to αVβ1 and α3β1 integrins on human corneal epithelial cells and that this interaction is important for infection. In contrast to viruses, membrane vesicles shed from Helicobacter pylori are heterogeneous in size and molecular composition. These vesicles harbour various adhesins and toxins that may facilitate binding to the cell surface and recruitment of different endocytic pathways. We developed a quantitative internalization assay and showed that the H. pylori vesicles were internalized mainly via clathrin-mediated endocytosis but were also capable of exploiting other endocytic pathways. Damage to the plasma membrane disrupts cellular homeostasis and can lead to cell death if not repaired immediately. Although endocytic mechanisms have been shown to be important for plasma membrane repair, little is known about their specific role. Listeriolysin O (LLO) is a bacterial toxin that can form pores in the plasma membrane and disrupt cellular homeostasis. We developed a reporter system for real-time imaging of the endocytic response to LLO pore formation. We found that two clathrin-independent endocytic pathways were important for plasma membrane repair. However, they were not directly involved in removing LLO pores from the plasma membrane. Our data suggests that these endocytic systems might rather influence membrane repair by their ability to regulate the plasma membrane composition, shape and tension. In conclusion, this thesis describes how pathogens and their virulence factors make use of specific mechanisms to enter host cells as well as revealing new insights on the role of the endocytic pathways in plasma membrane repair.