Ebola virus (EBOV) causes severe disease in humans characterized by high case fatality rates and significant immune dysfunction. A hallmark of EBOV infection is the formation of viral inclusions in the cytoplasm of infected cells. These inclusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV gene expression and genome replication, little is known about their role in the host response to infection. The cellular stress response is an antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2α). Related to this response is the post-transcriptional regulation of RNA mediated by stability elements called AU-rich elements (AREs) and their associated binding proteins (ARE-BPs), many of which are found in SGs. Because these processes have antiviral implications, many viruses have evolved strategies to interfere with SG formation, or appropriate ARE-BPs to benefit viral replication. However, it is unknown if EBOV interacts with these cellular systems. Here, we show that SG proteins were sequestered within EBOV inclusions where they formed distinct granules that colocalized with viral RNA. The inclusion-bound aggregates were not canonical SGs, and did not lead to translational arrest in infected cells. EBOV did not induce cytoplasmic SGs at any time post infection, but was unable to overcome SG formation induced by additional stressors. Despite the sequestration of SG proteins, canonical SGs did not form within inclusions. At high levels of expression, viral protein 35 (VP35), the viral polymerase co-factor that also mediates various immune evasion functions, disrupted SGs formation independently of eIF2α phosphorylation. Finally, we found that the cellular ARE-BP tristetraprolin (TTP) specifically targeted the 3’untranslated region (UTR) of the viral nucleoprotein (NP) mRNA and promoted its degradation. Interestingly, TTP was not found within viral inclusions, leading us to speculate that inclusions might serve to prevent viral RNA from encountering TTP. These results indicate that EBOV interacts with the cellular stress response and associated RNA regulatory proteins in ways that promote viral replication.

Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal was to identify NS2 residues important for interaction with the host ubiquitin system, as well as describe the mechanism by which NS2 induces host protein ubiquitination. Bioinformatics analysis provided a platform for development of loss-of-ubiquitin-function NS2 mutants. Combining critical mutations as double or triple NS2 ubiquitin mutants displayed an additive effect on reducing NS2-induced ubiquitination. Recombinant RSV (rRSV) containing NS2 ubiquitin mutations maintained their effect on ubiquitin expression during infection in addition to limiting STAT2 degradation activity. NS2 ubiquitin mutants decreased rRSV growth and increased levels of innate immune responses, indicating a correlation between NS2’s ubiquitin function and antagonism of type I IFN to enhance viral replication. Finally, several proteomics strategies were employed to identify specific cellular proteins ubiquitinated by NS2 to further define host-pathogen interactions during RSV infection. This study demonstrates an effective approach for limiting viral protein function to enhance immune responses during infection.

Les bactéries pathogènes intracellulaires manipulent les fonctions de la cellule hôte en sécrétant des facteurs de virulence (qu'on appelle effecteurs) dans le cytoplasme de la cellule infectée. Ce processus permet au pathogène de proliférer dans un environnement autrement hostile. L'identification et la caractérisation des effecteurs spécifiques des divers agents pathogènes est donc d'une importance cruciale pour contrer les infections bactériennes. Coxiella burnetii est un agent pathogène à Gram négatif de classe 3, responsable de la Fièvre Q, une zoonose qui entraîne des épidémies majeures, avec un fort impact sur l'économie et la santé. Les réservoirs naturels de Coxiella sont principalement les animaux d’élevage qui peuvent contaminer l’environnement en excrétant la bactérie principalement dans les produits de parturition, le mucus vaginal et les fèces. L’Homme s’infecte ensuite par inhalation de pseudo-spores disséminées dans l’environnement. La nature intracellulaire obligatoire de Coxiella a jusqu'ici sévèrement limité son étude et par conséquent, les facteurs de virulence bactériens impliqués dans le développement et la progression de l'infection restent encore largement inconnus. Coxiella se réplique à l'intérieur des cellules hôtes dans une grande vacuole présentant des caractéristiques autolysosomales. Le développement de la vacuole et la survie de Coxiella dans la cellule hôte sont dépendants de la translocation des effecteurs bactériens par un système de sécrétion de type 4 (SST4) Dot/Icm et de la manipulation de nombreuses voies de trafic et de signalisation de la cellule hôte par ces derniers. Notre équipe a généré et criblé la première banque de mutants par transposition de Coxiella, menant ainsi à l'identification d'un nombre important de potentiels déterminants de virulence et de protéines effectrices. Mon projet de thèse est basé sur la caractérisation de deux effecteurs de Coxiella, CvpF et AnkA, provenant de la banque de mutants générée par l’équipe. Les mutants de ces effecteurs présentent des phénotypes de défaut de réplication intracellulaire et de développement de vacuole. Ici, nous démontrons que l’effecteur CvpF est un substrat du système de sécrétion de type 4 Dot/Icm qui localise aux vacuoles contenant Coxiella (CCV). CvpF est également capable d'interagir avec Rab26, conduisant au recrutement du marqueur autophagosomal LC3B aux CCV. Les mutants de cvpF présentent un défaut de réplication in vitro et in vivo, suggérant que le détournement de l'autophagie par cet effecteur est crucial pour la virulence de Coxiella. Comme pour les mutants de cvpF, les mutants ankA présentent le même défaut de réplication in vitro et la protéine AnkA est un substrat du SST4. L'effecteur AnkA contient des motifs de répétition Ankyrin localisés sur son domaine N-terminal. La bactérie induit une hyperfusion des mitochondries de manière dépendante du SST4 et spécifique de l’effecteur AnkA. Nos résultats montrent que AnkA interagit avec Drp1, une protéine motrice impliquée dans la fission mitochondriale et que cette interaction ainsi que l’hyperfusion des mitochondries seraient dépendant du domaine contenant les répétitions Ankyrine. Le mécanisme par lequel AnkA agit sur Drp1 reste à déterminer. Cependant, les effets observés sur la mitochondrie suggèrent que la manipulation de l’organelle par la bactérie promeut le développement de la vacuole et la réplication intracellulaire du pathogène. En conclusion, notre recherche suggère fortement que de nombreux effecteurs de Coxiella manipulent les voies des cellules hôtes pour assurer le développement intracellulaire efficace de ce pathogène. / Intracellular pathogenic bacteria manipulate host cell functions by secreting virulence factors (known as effectors) into the cytoplasm of the infected cell. This process allows the pathogen to proliferate in an otherwise hostile environment. The identification and characterization of the specific effectors of the various pathogens is therefore of crucial importance to counteract bacterial infections. Coxiella burnetii is a Class 3 gram-negative pathogen that causes Q fever, a zoonosis that causes major epidemics with a high impact on the economy and health. The natural reservoirs of Coxiella are mainly farm animals that can contaminate the environment by excreting the bacteria mainly in parturition products, vaginal mucus and feces. Human is then infected by inhalation of pseudo-spores disseminated in the environment. The obligate intracellular nature of Coxiella has so far severely limited its study, and as result, bacterial virulence factors involved in the development and progression of infection remain largely unknown. Coxiella replicates within host cells in a large vacuole with autolysosomal characteristics. The development of vacuole and survival of Coxiella in the host cell depend on the translocation of bacterial effectors by the type 4 Dot / Icm secretion system (SST4B) and the manipulation of many trafficking and signaling pathways of the host cell. Our team has generated and screened the first library of Coxiella transposon mutants, leading to the identification of a significant number of candidate virulence determinants and effector proteins. My thesis project is based on the characterization of two effectors of Coxiella, CvpF and AnkA, from the mutant library generated by the team. Mutants of these effectors exhibit defect in intracellular replication and vacuole development phenotypes. Here, we demonstrate that the effector CvpF is a substrate of the SST4B that localizes to vacuoles containing Coxiella (CCV). CvpF is also able to interact with Rab26, leading to the recruitment of the LC3B autophagosomal marker to CCV. cvpF mutants exhibit in vitro and in vivo replication deficiencies, suggesting that diversion of autophagy by this effector is crucial for Coxiella virulence. As for cvpF mutants, ankA mutants show the same in vitro defect of replication and the protein AnkA is a substrate of the SST4. AnkA contains Ankyrin repetition patterns located on its N-terminal domain. The bacterium induces an AnkA-dependent hyperfusion of mitochondria. Our results show that AnkA interacts with Drp1, a motor protein involved in mitochondrial fission, and that this interaction as well as mitochondrial hyperfusion is dependent on the domain containing Ankyrin-repeat motifs. The mechanism by which AnkA acts on Drp1 remains to be determined. However, the observed effects on mitochondria suggest that the organelle's manipulation by the bacterium promotes the development of the vacuole and the intracellular replication of the pathogen. To conclude, our research strongly suggests that multiple Coxiella effectors manipulate host cell pathways to ensure the efficient intracellular development of this pathogen.

Host metabolism is profoundly altered during bacterial infection, both as a consequence of immune activation and secondary to virulence strategies of invading pathogens. As a result, the metabolic pathways that regulate nutrient acquisition, energy storage, and resource allocation in host cells must adapt to pathogen stress in order to meet the physiological demands of the host during infection. In this work, we uncover that the synthesis of the monounsaturated fatty acid (MUFA) oleate is necessary for the pathogen-mediated induction of immune defense genes. Accordingly, C. elegans deficient in oleate production are hypersusceptible to infection with diverse human pathogens, which can be rescued by the addition of exogenous oleate. However, oleate is not sufficient to drive protective immune activation. Oleate is also important for proper lipid storage and abundance. We found that exposure to pathogenic bacteria drives rapid somatic depletion of lipid stores in C. elegans. Activating the p38/MAPK immune signaling pathway in the absence of pathogens was also sufficient to drive loss of somatic fat. In addition, we found that transcriptional suppression of MUFA synthesis occurs during P. aeruginosa infection, in a manner dependent on pathogen virulence. Finally, we showed that the host compensates for the pathogen-induced depletion of fatty acids by promoting the redistribution of oleate from non-intestinal tissues to support immune function in the intestine. Together, these data add to the known health-promoting effects of MUFAs, and suggest an ancient link between nutrient stores, metabolism, and host responses to bacterial infection.

Recognition ofmicrobial products by germ-line-encoded PRR initiates immune responses, but how PRR mediate specific host responses to infectious agents is poorly understood. We and others have proposed that specificity is achieved by collaborative responsesmediated between different PRR. One such example comprises the fungal β-glucan receptor Dectin-1, which collaborates with TLR to induce TNF production. We show here that collaborative responses mediated by Dectin-1 and TLR2 are more extensive than first appreciated, and result in enhanced IL-23, IL-6 and IL-10 production in DC, while down-regulating IL-12 relative to the levels produced by TLR ligation alone. Such down-regulation occurred with multiple MyD88-coupled TLR, was dependent on signaling through Dectin-1 and also occurred in macrophages. These findings explain how fungi can induce IL-23 and IL-6, while suppressing IL-12, a combination which has previously been shown to contribute to the development of Th17 responses found during fungal infections. Furthermore, these data reveal how the collaboration of different PRR can tailor specific responses to infectious agents.

Ebola virus (EBOV) and other members of the Filoviridae family are enveloped RNA viruses that are the causative agents of sporadic outbreaks of highly lethal disease in humans and non-human primates. EBOV entry into host cells requires attachment, internalization, and subsequent trafficking to the late endosomal/lysosomal compartment in order to reach the filovirus entry receptor, Niemann-Pick C1 (NPC1) and other triggering factors required for EBOV glycoprotein (GP)-mediated fusion between the viral and host membranes. The highly regulated nature of endosomal trafficking coupled with the dependence of EBOV on accurate endolysosomal trafficking for entry led us to hypothesize that the virus depends on—and potentially actively regulates—a consortium of specific host trafficking factors. In this thesis, we investigated the role of two trafficking complexes involved in endosomal maturation and trafficking, the Homotypic Fusion and Vacuole Protein Sorting (HOPS) complex and the PIKfyve-ArPIKfyve-Sac3 (PAS) complex, in EBOV entry. Furthermore, in order to further dissect how the PAS complex is regulated and performs its effector functions, we performed a protein-protein interaction screen using BioID in order to define the PAS cellular interactome. Using an inducible CRISPR/Cas9 system, we found that depletion of each HOPS subunit, as well as depletion of a positive regulator of the HOPS complex, UVRAG, impaired EBOV entry. Furthermore, we mapped a region of UVRAG spanning residues 269-442 to be key for binding to the HOPS complex and mediating EBOV entry, indicating that expression of and coordination between the HOPS complex and UVRAG are required for EBOV entry. Similarly, knockout of each subunit of the PAS complex was found to impair EBOV entry. Further molecular dissection using small molecule inhibitors and enzymatic mutants of PIKfyve and Sac3 demonstrated that PIKfyve kinase activity is required for EBOV entry, while Sac3 phosphatase activity is dispensable. Using a fluorescent probe for phosphatidylinositol(3,5)bisphosphate, the lipid product generated by PIKfyve, we also found evidence that stimulation of cells by EBOV virus-like-particles enhances PIKfyve activity, suggesting that the virus can promote its entry by activating the PAS complex. Finally, using BioID to screen for interacting proteins of the PAS complex, we identified candidate interactors involved in endosomal trafficking as well as other cell processes including mitochondrial function and cell cycle regulation. Further characterization of one candidate interactor, the coatomer complex I (COPI), using proximity ligation assays validated the interaction between ArPIKfyve and COPI subunit COPB1, and provides further evidence for a role of COPI in endosomal trafficking. Taken together, these results highlight the importance of cellular trafficking factors involved in diverse facets of endosomal dynamics, from lipid metabolism to membrane tethering, for the entry of EBOV and other filoviruses, and further shed light on how EBOV can actively modulate host trafficking networks to promote successful viral entry and infection. Further molecular dissection of how the virus hijacks cell trafficking will facilitate the development of antiviral therapeutics as well as elucidate how these fundamental cellular processes are regulated.

No description available.

No description available.

No description available.

Burkholderia pseudomallei is an opportunistic soil pathogen that causes melioidosis, a life-threatening human disease prevalent in Southeast Asia, northern Australia, the Middle East, Africa, and South America. The organism also causes disease in plants and animals. Persons with severe melioidosis usually die of septicemia. Relatively little is known regarding the virulence mechanisms of B. pseudomallei; however, several putative virulence determinants have been identified. The organism is able to invade and replicate within phagocytic cells and is particularly pathogenic in males with diabetes mellitus. B. thailandensis is closely related to B. pseudomallei, but is not pathogenic. This study examines various in vitro monocyte / macrophage infection models used to study innate immune responses to B. pseudomallei. Several monocyte and macrophage models showed little or no significant differences between proinflammatory cytokines secreted in response to infection with B. pseudomallei and B. thailandensis. Peripheral blood monocytes from diabetic males produced lower normalized levels of proinflammatory cytokines IL-1α, IL-1β, IL-6, and IL-8 than monocytes from healthy males in response to infection with B. pseudomallei, B. thailandensis, and E. coli. Surprisingly, normalized levels of secreted IL-1β from B. pseudomallei-infected monocytes from diabetic females were higher than levels from healthy females. The results revealed a significant interactive effect of gender and diabetic state on peripheral blood monocyte secretion of IL-1β (p = 0.0370) and IL-8 (p = 0.0390), as well as a significant interactive effect of diabetic state and type of infectious agent on peripheral blood monocyte secretion of IL-1α (p=0.0210) and IL-6 (p=0.0204). These results may help explain why diabetic males are unusually susceptible to infection with B. pseudomallei.