6 Abstract This master's thesis provides study of individual helixes from C-terminal pore-forming domain (CTD) of colicin U and their behavior in lipid bilayer on atomic level. For this purpose the all-atom molecular simulation method was used. Later the study was extended an applied on CTD of published structures of other pore-forming colicins. On the base of study extension the ability of disruption of lipid bilayer integrity by helixes H1 and H10 was successfully observed. Helix H1 was synthesized and its activity was experimentally proved on black lipid membranes. The other helixes are often too short to be able to keep position in lipid bilayer and their behavior could be affected by artificial termini, therefore they were not synthesized. The MD simulations of pairs of helixes show that structure stability and their ability to stay in the membrane depends on binding partners. The results of the thesis show the importance of H10 for colicin pore-formation, which has not been observed yet. The results also support the toroidal pore model suggested previously for colicin E1. The results prove that colicins contain specific secondary structures, which are able to disrupt the inner bacterial membrane not only in its native form but also when artificially separated from the rest of the protein. Klíčová...

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier. / Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment.

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Cry6Aa1, une nouvelle toxine produite par Bacillus thuringiensis (Bt), agit comme insecticide sur la chrysomèle du maïs (WCRW). Dans cette étude, on démontre que Cry6Aa1 est une toxine formeuse de pores (TFP) en bicouches lipidiques planes (BLP). Contrairement aux autres toxines de Bt étudiées jusqu’à présent, la formation de pores par Cry6Aa1 ne requiert pas de prétraitement par protéases et se produit à des doses de toxine deux à trois ordres de grandeur plus faibles que celles nécessaires pour les autres toxines de Bt dans les mêmes conditions. La formation de pores par la forme non traitée de Cry6Aa1 dépend du pH; les pores obtenus ont des conductances comprises entre 31 et 689 pS en conditions symétriques de 150 mM de KCl; ils sont cationiques avec un comportement cinétique complexe. Les propriétés biophysiques des pores ne changent pas lorsque la toxine est traitée avec le suc du mésenthéron de l’insecte (Cry6Aa1 WCR1). Par contre, un traitement à la trypsine (Cry6Aa1 TT) modifie la conductance et la sélectivité des pores à pH 5,5 (le pH physiologique de l’intestin de WCRW). La reconstitution en BLP de fraction de membrane native du mésenthéron de WCRW affecte les propriétés des pores formés par Cy6Aa1. Les déterminants moléculaires du mode d’action de cette nouvelle toxine formeuse de pores semblent donc différer de ceux décrits précédemment pour d’autres toxines de Bt. La structure atomique tridimensionnelle de Cry6Aa1 vient tout juste d’être élucidée. Elle montre que la toxine adopte une conformation riche en hélices α qui ressemble fortement à celle de la TFP ClyA produite par E. coli. En se fondant sur les données disponibles pour ClyA, on a étudié l’effet de divers changements dans les régions N et C terminales de Cry6Aa1 sur sa capacité de former des pores en BLP. / Cry6Aa1 is a new toxin produced by Bacillus thuringiensis (Bt), which displays insecticidal activity against the Western corn rootworm (WCRW). The present work demonstrates that Cry6Aa1 is a pore-forming toxin (PFT) in planar lipid bilayers (PLBs). Contrary to other Bt toxins tested so far, pore formation by Cry6Aa1 does not require protease pretreatment and takes place at doses that are two to three orders of magnitude lower than those required for other Bt toxins under similar conditions. Pore formation by Cry6Aa1 is pH-dependent; the conductances of the pores range between 31 and 689 pS under symmetrical 150 mM KCl conditions; they are cationic and display a complex kinetic behaviour. The treatment of the toxin with midgut juice (Cry6Aa1 WCR1) does not change the biophysical properties of the pores. However, the treatment with trypsin (Cry6Aa1 TT) affects their conductance and selectivity at pH 5.5 (the WCRW gut physiological pH). The incorporation in PLBs of native membrane material from WCRW midgut affects the behaviour of the Cry6Aa1 pores. The molecular determinants of the mode of action of this new PFT appear therefore to differ from those reported before for other Bt toxins. The three-dimensional (3-D) atomic structure of Cry6Aa1 has just been elucidated. It shows that the toxin assumes an α-helix-rich configuration, which is quite similar to that of the ClyA PFT produced by E. coli. Based on the data available for ClyA, we have studied how different changes in the N- and C-terminal regions of Cry6Aa1 affect its pore formation ability in PLBs.

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.

Of the eleven species of bacteria that comprise the genus Yersinia of the family Enterobacteriaceae, three species are pathogenic for humans. Yersinia pseudotuberculosis and Yersinia enterocolitica usually cause a mild, self-limiting mesenteric lymphadenitis or ileitis. Yersinia pestis causes a highly invasive often fatal disease known as plague. All three elaborate a type three secretion system that is essential for virulence and encoded on closely related plasmids. In Y. pestis, all the effectors, structural components and chaperones are encoded on the 70kb plasmid, pCD1. Of these, LcrV from Y. enterocolitica has been implicated in playing an immunosuppressive role through its interaction with host Toll-like receptor 2 (TLR2) and induction of IL-10. Through expression and purification of recombinant LcrV from Escherichia coliwe show that only high molecular weight species of rLcrV are able to stimulate TLR2. In a highly sensitive subcutaneous mouse infection model we demonstrate no difference in the time to death between TLR2-sufficient or deficient mice. Analysis of cytokine levels between these two genotypes also shows no significant difference between splenic IL-10 and IL-6 or levels of bacteria. We conclusively show that this interaction, if it does occur, plays no significant role in vivo. In a separate set of experiments, we also determined that the expression of F1, a peptide shown to be responsible for 37°C-dependent inhibition of invasion by Y. pestis in vitro, was significantly decreased under high oxygen conditions. This led us to re-examine the invasion phenotype both in vitro and in vivo. These results give new insights into virulence gene expression in Y. pestis by environmental cues other than temperature.

Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.