<p>Guanylate binding proteins (Gbps) are a family of large GTPases that are highly stimulated by IFNγ and confer resistance to various viral, protozoan, and bacterial pathogens. Following infections of intracellular pathogens, multiple Gbps can localize to pathogen vacuoles and promote the vesiculation and destruction of these structures. While Gbps have also been implicated in pathways independent of vacuolar disruption, their roles in these processes have been less characterized. In this dissertation, I focus on the mechanism of Gbps downstream of vacuolar disruption in order to further elucidate the role of these proteins during immune responses. </p><p> Due to the IFNγ stimulation of caspase-11 pyroptosis, I first addressed the ability of Gbps to promote the non-canonical caspase-11 dependent pathway of pyroptosis. I found that Gbpchr3-/- cells had reduced cell death in response to the vacuolar pathogen, L. pneumophila, and various LPS ligands. Using YFP-Gal3 as a marker for damaged membranes, I showed that there were equivalent levels of damaged pathogen vacuoles between WT and Gbpchr3-/- cells suggesting these proteins promoted pyroptosis independently of vacuolar disruption. Instead, it appears that Gbps modulate the activation of caspase-11 following LPS release into the cytosol. </p><p> The recruitment of Gbps is mediated by multiple host proteins including the Immunity Related GTPases and the autophagy conjugation system. I found in the second study that at least one Gbp, Gbp2, was also recruited to damaged vacuoles through the aid of Galectin-3, a β-galactoside binding protein, as well as the autophagy adaptor protein, p62. As all three proteins were also recruited to sterile damaged vesicles created by hypotonic shock, calcium phosphate precipitates, and lysosomal damage, it suggests Gbps are recruited through a universal mechanism which is independent of PAMP recognition. Interactions between p62, Gbp2, and Gal3 present a model whereby p62 facilitates the recruitment of Gbp2 to damaged membranes through interactions with Galectin-3. Their localization to these sites may subsequently facilitate autophagic degradation of membranes or promote the recruitment of pyroptotic complexes to modulate immune functions although this remains to be elucidated. </p><p> This dissertation examines the less characterized roles of Gbps downstream of vacuolar disruption. By uncovering these alternative pathways, this work provides a foundation to study the variations within the Gbp family and allows for the field to further understand the mechanisms by which they promote cellular immune responses.</p> / Dissertation

Francisella tularensis, l'agent de la tularémie, est une bactérie intracellulaire capable d'infecter un grand nombre de cellules dont les macrophages. Le système immunitaire inné cytosolique est capable de détecter la bactérie à différents stades de son cycle d'infection. Dans un premier temps, le macrophage détecte la bactérie cytosolique et produit de l'interféron de type I. Cet interféron induit l'expression de milliers de gènes. Le macrophage est ensuite capable de détecter l'ADN cytosolique de la bactérie via un récepteur spécifique AIM2. La liaison AIM2-ADN entraine la formation d'un complexe multi-protéique appelé inflammasome et se composant de AIM2-ASC-caspase-1. L'activation de ce complexe conduit à la maturation de la caspase-1. Caspase-1 permet la sécrétion de deux cytokines majeures antimicrobiennes : l'IL-1beta et l'IL-18. De plus, caspase-1 induit une mort programmée des cellules infectées appelée pyroptose. La sécrétion de cytokines et la pyroptose sont deux évènements majeurs pour lutter contre les pathogènes. Ma thèse a consisté à identifier le lien entre l'interféron et l'activation de l inflammasome AIM2 dans des macrophages infectés par la bactérie Francisella. En réalisant un crible a l'aide d'ARNs interférents, j'ai découvert que 2 protéines sont impliquées dans l'activation de cet inflammasome, les guanylate binding proteins 2 et 5 (GBP2 et GBP5). En collaboration avec l'équipe du Dr. Broz en Suisse, nous avons démontré que les GBPs étaient impliquées dans le contrôle de la réplication intracellulaire de Francisella et également dans la lyse de la bactérie permettant le relargage d'ADN et l'activation de l'inflammasome AIM2. Les GBPs sont induites par l'interféron de type I mais très majoritairement par l'interféron de type II (IFN- gamma). Nous avons mis en évidence que le contrôle de la réplication bactérienne est GB dépendant et inflammasome-dépendant en absence d'IFN- gamma mais qu'il devient totalement GB dépendant et inflammasome-indépendant dans des macrophages pré-stimulés avec de l'IFN- gamma. De plus, la mort des macrophages pré-stimulés avec de l'IFN- gamma et infectés par Francisella est également GBP-dépendante et inflammasome-indépendante. En prenant en compte tous ces résultats, nous concluons que les GBPs sont des protéines impliquées dans l'immunité des macrophages infectés par Francisella mais qu'elles ont un double rôle : d'une part celui d'induire l'activation de l'inflammasome (la pyroptose) sous le contrôle de l'interféron de type I et d'autre part, d'induire une mort cellulaire et la lyse des bactéries cytosoliques de manière indépendante de l'inflammasome sous le contrôle d'IFN- gamma. Nos résultats placent donc les GBPs comme les effecteurs majeurs de l'immunité cytosolique antibactérienne suite au traitement par l'IFN-gamma / Francisella tularensis is an intracellular bacterium, and the causative agent of tularemia, capable of infecting a large number of cells including macrophages. The innate cytosolic immune system is capable of detecting the bacterium at different stages of its infection cycle. Macrophages first detect the DNA of the cytosolic bacterium and produce type I interferon. Type I interferon subsequently induces the expression of thousands of genes. The macrophages then detect the cytosolic DNA of the bacterium via a cytosolic DNA sensor called AIM2. The AIM2-DNA binding results in the formation of a multi-protein complex called the AIM2 inflammasome composed of AIM2-ASC-caspase-1. Activation of this complex leads to the maturation of caspase-1. Caspase-1 activation leads to the secretion of two major antimicrobial cytokines, IL-1ß and IL-18. In addition, caspase-1 induces a programmed cell death termed pyroptosis. Cytokine secretion and pyroptosis are two major events in the control of pathogens. My PhD focused in identifying the link between interferon and activation of the AIM2 inflammasome in macrophages infected with the pathogenic bacterium Francisella. I performed a RNA interference screening and identified two proteins involved in the activation of the AIM2 inflammasome: guanylate binding proteins 2 and 5 (GBP2 and GBP5). In collaboration with Dr. Broz’s team in Switzerland, we demonstrated that GBPs are involved in the control of intracellular replication of Francisella and also in the lysis of the bacterium allowing the release of bacterial DNA and the activation of inflammasome AIM2. GBPs are induced by type I interferon but to a much greater extent by type II interferon (IFN-gamma). In the second part of my work, we demonstrate that the control of bacterial replication is GBP-dependent and inflammasome-dependent in the absence of IFN-gamma but that it becomes fully GBP-dependent and inflammasome-independent in macrophages primed with IFN-gamma. Cell-death of macrophages primed with IFN-? and infected with Francisella is also GBP-dependent and inflammasome-independent. Taken together, these results demonstrate that GBPs are innate immunity proteins involved in the death of macrophages and the bacterial growth restriction through two differents pathways : one induces the activation of inflammasome (induction of Pyroptosis) controlled with type I interferon signaling and, another induces cell-death and bacterial killing in an inflammasome-independent manner under the control of IFN-gamma. Our results thus discriminates the antimicrobial action of the inflammasome and of GBPs and position GBPs as the master antibacterial effectors of IFN-gamma, a key cytokine to fight cytosolic bacteria

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