Thesis (Ph.D.)--Murdoch University, 2009. / Thesis submitted to the Faculty of Health Sciences. Includes bibliographical references (p. 246-272)

Indiana University-Purdue University Indianapolis (IUPUI) / Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrate that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.

L’objectif de cette thèse a été d’enrichir nos connaissances sur les bactériesintracellulaires strictes et spécialement Coxiella burnetii, agent responsable de la fièvre Q.Pour ce faire, nous avons d’une part amélioré les techniques de préparation de l’ARN pourles études transcriptionnelles et d’autre part utilisé la technologie des puces à ADN pouranalyser le transcriptome ainsi que la diversité génomique de C. burnetii.L’utilisation d’échantillons cliniques dans les études transcriptionnelles est limitéepar la quantité de matériel disponible qui ne permet pas d’analyser simultanément les profilsdu pathogène et de son hôte au cours de l’infection. De ce fait, nous avons développé, sur unmodèle d’escarres obtenus à partir de malades de fièvre boutonneuse méditerranéenne, unestratégie basée sur l’hybridation soustractive pour séparer les ARN eucaryotes etprocaryotes dans le but d’entreprendre des hybridations de puces à ADN.C. burnetii est une bactérie hautement résistante aux stress environnementaux commele changement de pH, la dessiccation, mais aussi le changement de température. Nous avonsparticulièrement étudié la réponse précoce de C. burnetii lors d’une exposition à une hauteet faible température. L’analyse globale du profil transcriptionnel a montré que la réponsede C. burnetii était limitée et similaire pour les différents stress appliqués. Cependant,malgré cette faible réponse, il apparait clairement qu’une accumulation de ppGpp, un arrêtde la croissance et des modifications de la membrane et de la paroi cellulaire permettraient àC. burnetii de résister à ces stress. Toutes ces régulations géniques convergent vers unchangement d’état de la bactérie vers une forme pseudo-sporulée. De plus, nous avonsobservé une organisation spatiale des gènes différentiellement exprimés. Nos analyses bioinformatiquesont montré que ces clusters de régulation ne répondaient ni au paradigmepromoteur - facteur de transcription – opéron, ni à des réseaux biologiques. Ayant retrouvéce phénomène dans plusieurs autres études transcriptionnelles chez d’autres bactériesintracellulaires, nous spéculons que ces clusters de régulations pourraient être dus à unerégulation épigénétique qu’il reste à caractériser.Différentes méthodes de typage ont déjà été mises au point pour classer les isolats deC. burnetii dans le but d'explorer son pouvoir pathogène. Ici, nous présentons une méthodede génomotypage basée sur la présence ou l'absence de gènes à l'aide de puces à ADN.Nous avons testé notre stratégie de génomotypage sur 52 isolats provenant de différenteszones géographiques, de différents hôtes et de patients présentant différentes manifestationscliniques. L'analyse a révélé la présence de 10 génomotypes organisés en 3 groupes avecune topologie congruente à celle observée avec le Multi Spacer Typing. Nous avons aussidécouvert 4 génomotypes particulièrement associés à la fièvre Q aiguë, alors que tous lesgénomotypes étaient associés à la forme chronique. De plus, le génomotypage a révélé queles isolats retrouvés dans les tiques dures, y compris la souche de référence Nine Mileappartiennent au même genomotype.Globalement, les données que nous avons obtenues confirment le fait que les puces àADN sont un outil adapté pour l’analyse de la pathogénicité de C .burnetti mais aussi desautres bactéries intracellulaires strictes. Cependant, de nouvelles technologies plusrésolutives comme le DNA ou RNAseq semblent être plus prometteuses mais restent encoreà optimiser / The objective of this thesis was to increase knowledge of obligate intracellular bacteriaand specifically, the causative agent of Q fever C. burnetii. In this regard, we have improvedstrategies to purify RNA for the transcriptional studies. We also used the technologymicroarrays to analyze the transcriptome and genomic diversity of C. burnetii.The use of clinical samples in the transcriptional studies is limited by the amount ofmaterial available and thus the transcriptional profiles of the pathogen and its host duringinfection can not be simultaneously analyze. We developed, with a model of eschars obtainedfrom Mediterranean spotted fever patients, a strategy based on subtractive hybridization toseparate RNA eukaryotic and prokaryotic cells in order to perform microarray experiments.Analysis of the survival strategies used by this bacterium to adapt to new environmentalconditions is critical for our understanding of C. burnetii pathogenicity. Here, we report theearly transcriptional response of C. burnetii under temperature stresses. Our data show thatC. burnetii exhibited minor changes in gene regulation under short exposure to heat or coldshock. While small differences were observed, C. burnetii seemed to respond similarly to coldand heat shock. The expression profiles obtained using microarrays produced in-house wereconfirmed by quantitative RT-PCR. Under temperature stresses, 190 genes were differentiallyexpressed in at least one condition, with a fold change of up to 4. Globally, the differentiallyexpressed genes in C. burnetii were associated with bacterial division, (p)ppGpp synthesis,wall and membrane biogenesis and, especially, lipopolysaccharide and peptidoglycansynthesis. These findings could be associated with growth arrest and witnessed transformationof the bacteria to a spore-like form. Unexpectedly, clusters of neighboring genes weredifferentially expressed. These clusters do not belong to operons or genetic networks; theyhave no evident associated functions and are not under the control of the same promoters. Wealso found undescribed but comparable clusters of regulation in previously reportedtranscriptomic analyses of intracellular bacteria, including Rickettsia sp. and Listeriamonocytogenes. The transcriptomic patterns of C. burnetii observed under temperature stressespermits the recognition of unpredicted clusters of regulation for which the trigger mechanismremains unidentified but which may be the result of a new mechanism of epigenetic regulation.Different typing methods have been previously developed to classify C. burnetii isolatesin order to explore its pathogenicity. Here, we report a comprehensive genomotyping methodbased on presence or absence of genes using microarray. The genomotyping method was thentested on 52 isolates obtained from different geographic areas, different hosts and isolated frompatient with different clinical manifestations. The analysis reveals the presence of10 genomotypes organized in 3 groups with a topology congruent with that of Multi SpacerTyping. We also found out 4 genomotypes especially associated with acute Q fever whereas allthe genomotypes could be associated to chronic human infection. Serendipity, genomotypingreveals that hard ticks isolates including Nine Mile belong to the same genomotype.Overall, the data we obtained confirm that DNA microarrays are a suitable tool forexploring pathogenicity of C. Burnetti and other obligate intracellular bacteria. However newtechnologies such as DNAseq or RNAseq seem more promising but still need to optimize andalso are still expensive compared to microarray.

A travers l'évolution, les agents pathogènes ont développé des stratégies leur permettant de survivre au sein de leur hôte en interférant avec la biogénèse des phagolysosomes. Comme C. burnetii vit dans un phagosome acide incapable de fusionner avec les lysosomes et que sa virulence est associée à l'expression de son LPS, nous avons étudié le rôle du LPS de C. burnetii dans le détournement de la conversion phagosomale. En effet, nous avons montré que C. burnetii virulent ainsi que son LPS se localisent dans des compartiments Lamp-1+ qui n'acquièrent pas la CathepsineD et Rab7 n'est pas recruté à leur surface. Contrairement au LPS du variant avirulent de C. burnetii, qui est localisé dans les lysosomes, le LPS de C. burnetii virulent (vLPS) n'induit pas l'activation de la MAPKinase p38, empêche le recrutement de Rab7 à la surface des compartiments en déstabilisant le complexe HOPS. Finalement, nous avons démontré que la bactérie virulente exprimant le vLPS détourne la conversion phagosomale pour survivre et pour se multiplier dans les macrophages en évitant l'activation de l'axe MAPK-p38/Vps41-HOPS. Nous avons également étudié les mécanismes permettant à Tropheryma whipplei, l'agent de la maladie de Whipple, de se répliquer dans les macrophages puisque les macrophages sont la cible in vivo de cette bactérie. Nous avons montré que T. whipplei bloque la conversion de son phagosome. / Through evolution, pathogens have developed strategies to survive within their host by interfering with the biogenesis of phagolysosomes. As it is known that C. burnetii lives in acidic phagosome which is unable to fuse with the lysosomes and that its virulence is associated with the expression of LPS, we studied the role of C. burnetii LPS in hijacking of phagosomal conversion. Indeed, we showed that the virulent C. burnetii and its LPS are located in Lamp-1+ compartments which do not acquire CathepsineD and Rab7 is not recruited to their surface. Contrary to LPS of avirulent C. burnetii, which is located in the lysosomes, the LPS of virulent C. burnetii (vLPS) does not induce activation of the p38 MAPKinase and it prevents the recruitment of Rab7 to the surface of compartments by destabilizing the HOPS complex. Finally, we demonstrated that virulent bacteria expressing virulent LPS hijack phagosomal conversion to survive and multiply in macrophages by preventing activation of p38-MAPK/Vps41HOPS axis. We also studied the mechanisms by which Tropheryma whipplei, the agent of Whipple's disease, replicates in macrophages, which are its target in vivo. We have shown that T. whipplei blocks the conversion of its phagosome. Indeed, after purification of phagosomes containing-T. whipplei, we observed by Western blot and confocal microscopy that T. whipplei survives in an immature phagosome with characteristics of both early and late phagosomes (presence of Rab5 and Rab7) making it unable to fuse with lysosomes. As the IL-16 is known to induce replication of T. whipplei, we studied the effect of this cytokine on the phagosome biogenesis of T. whipplei.

Le nombre de nouvelles espèces bactériennes identifiées augmente constamment. L'un des facteurs les plus importants de cette situation est l'approche culturomique. Par conséquent, la nécessité de classifier et de décrire taxonomiquement ces microorganismes pour comprendre leur évolution, leurs caractéristiques et leurs relations avec d'autres organismes vivants a émergé. À ce jour, plus de 170000 génomes bactériens sont disponibles. Face à cette situation, la taxonomie bactérienne ne peut être éloignée des informations fournies par le génome entier. La stratégie taxonogénomique a été élaborée dans notre laboratoire pour inclure les séquences du génome dans le schéma taxonomique. Cette nouvelle stratégie consiste en la combinaison de diverses données phénotypiques, et l'analyse génomique et la comparaison, aboutissant à une description rationnelle et complète de nouveaux taxons bactériens. Un deuxième aspect de notre travail de thèse a été l'analyse pangénomique de Coxiella burnetii. Profitant de la disponibilité de 75 génomes de C. burnetii, nous avons montré que ce pangénome est ouvert, ce qui s’oppose à ceux des autres bactéries intracellulaires. Aucun contenu génique spécifique à une maladie, ou à un géovar spécifique n'a pu être identifié. En revanche, l'analyse phylogénétique basée sur le core génome correspondait à celle obtenue à partir du typage. Ainsi, nous démontrons ici que le séquençage du génome peut être bien adapté à la description officielle ainsi que l'analyse pangénomique des bactéries associées à l'homme, et permettre de répondre à de nombreuses questions microbiologiques telles que l'évolution, la résistance aux antibiotiques et la pathogénicité. / The number of new identified bacterial species is constantly increasing. One of the most important contributors to this situation is the microbial culturomics approach. Consequently, the need to classify and taxonomically describe those microorganisms to understand their evolution, characteristics and relationships with other living organisms has emerged. To date, more than 170000 bacterial genomes are available. Facing this situation, bacterial taxonomy cannot be disconnected from the information provided from the whole genome. The taxono-genomics strategy was elaborated in our laboratory to include genome sequences in the taxonomic scheme. This new strategy consists in the combination of various phenotypic data and genomic analysis and comparison, resulting in a rational and comprehensive description of new bacterial taxa. A second aspect of our thesis work was the pangenomic analysis of Coxiella burnetii. Taking advantage of the availability of 75 C. burnetii genomes, we showed that this pangenome is open, which contrasts with those of other intracellular bacteria. No disease-specific, or geovar-specific gene content could be identified. In contrast, the core gene-based phylogenetic analysis matched that obtained from multi-spacer typing. Thus, we herein demonstrate that genome sequencing may, among its many applications, be well suited for the official description as well as pangenome analysis of human-associated bacteria, including pathogens, and may allow addressing many microbiological questions, such as evolution, outbreaks, antibiotic resistance, and pathogenicity.

Coxiella burnetii é a bactéria intracelular causadora da Febre Q, capaz de subverter funções celulares e evadir o reconhecimento do sistema imune da célula hospedeira permitindo o estabelecimento do seu nicho replicativo nas células-alvo: macrófagos e monócitos. É um patógeno altamente virulento, sendo necessário poucos organismos para desencadear a doença, e é considerado como um potencial agente de bioterrorismo da categoria B. Entre os danos econômicos causados por C. burnetii destaca-se a infecção de animais de gado, seu reservatório natural, pois causa aborto espontâneo dos filhotes e, consequentemente, prejuízo aos produtores. Seres humanos também adquirem a infecção, por inalação de partículas contaminadas. Hospedeiros imunocompetentes são capazes de restringir a infecção por C. burnetii apesar dos diversos mecanismos de evasão da resposta imune do hospedeiro, como interação com vias de sinalização celular e utilização de efetores bacterianos. No entanto, em hospedeiros não competentes a infecção pode evoluir para casos de Febre Q crônica e levá-los à morte. Modelos murinos são frequentemente utilizados para entender as interações patógeno-hospedeiro nas infecções por essa bactéria. A diferença na suscetibilidade de macrófagos de diferentes linhagens murinas e de diferentes tipos celulares à infecção por C. burnetii ainda é pouco conhecida. No entanto, sabe-se que C. burnetii fase II sucumbe a macrófagos derivados da medula óssea (BMDMs) de camundongos C57BL/6, enquanto células das linhagens BALB/c e A/J são suscetíveis à infecção. Nesse contexto, considerando a relevância biomédica de C. burnetii, faz-se necessário a determinação de um modelo relevante para melhor compreender as relações patógeno-hospedeiro nas infecções por essa bactéria. Neste trabalho, caracterizamos um novo modelo de estudo com macrófagos primários que permite avaliar a infecção com C. burnetii fase II in vitro, além de elucidar os mecanismos relacionados à suscetibilidade das células à bactéria. Por meio de quantificação do DNA genômico bacteriano por qPCR verificamos que macrófagos alveolares (AMs) murinos são altamente suscetíveis à replicação da bactéria, mesmo no fundo gênico restritivo C57BL/6. Caracterizamos a replicação da bactéria em AMs por microscopia de fluorescência e microscopia eletrônica de transmissão e demonstramos que essa replicação ocorre dentro dos vacúolos parasitóforos típicos. Pela análise de expressão gênica por RT-PCR efenotipagem de marcadores de superfície por FACS identificamos que a alta suscetibilidade dos AMs se deve a uma polarização dessas células para um padrão M2. Por fim, validamos a relevância desse modelo pela análise da suscetibilidade de AMs murinos deficientes para NOS2, IFN-? e IL-4 como prova de princípio. Verificamos que a suscetibilidade de AMs é comparável à de células Vero e células THP-1, modelos celulares imortalizados descritos como altamente suscetíveis à replicação de C. burnetii, e que AMs podem ser utilizados para estudos utilizando células derivadas de camundongos com fundo gênico C57BL/6. Dessa forma, nosso trabalho caracterizou os AMs murinos como um relevante modelo celular primário altamente suscetível para o estudo das interações patógeno-hospedeiro frente à infecção in vitro por C. burnetii fase II, além de elucidar os mecanismos por trás dessa suscetiblidade / Coxiella burnetii is the intracellular bacterium that causes Q fever, capable of subverting cellular functions and evading recognition by the host cell immune system, thus allowing the establishment of its replicative niche in its the target cells: macrophages and monocytes. It\'as a highly virulent pathogen, with few organisms being sufficient to cause the disease, and considered a potential class B bioterrorism agent. Among the economic damages caused by C. burnetii are infection of cattle animals, its natural reservoir, that leads to spontaneous abortion of the offspring and financial loss to the producers. Human beings also acquire the infection, through inhalation of contaminated air particles. Immunocompetent hosts are able to restrict infection by C. burnetii despite the several evasion mechanisms from the host immune system, which includes interaction with cell signaling pathways and utilization of bacterial effectors. However, in non-competent hosts the infection can evolve to chronic Q Fever and lead to death. Murine models are frequently used to understand the host-pathogen interactions during infections by this bacterium. The difference in the susceptibility among macrophages from different murine strains, as well as different cell types, still poorly known. However, it is known that C. burnetii phase II succumbs to bone-marrow derived macrophages (BMDMs) from C57BL/6 mice, whereas cells from BALB/c and A/J strains are susceptible to infection. In this context, considering the biomedical relevance of C. burnetii, it\'s necessary to establish a relevant study model to better understand the host-pathogen relations in infections by C. burnetii. In this work, we characterized a new study model with primary macrophages to assess the infection by C. burnetii phase II in vitro and elucidated the mechanisms underlying the susceptibility to C. burnetii. By using qPCR for quantification of bacterial genomic DNA, we saw that murine alveolar macrophages (AMs) are highly susceptible to C. burnetii replication, even in the usually restrictive C57BL/6 backgound. We characterized the bacterium replication in murine AMs by florescence microscopy and transmission electron microscopy, showing that this replication occurred inside the typical C. burnetii-containing vacuole. We also identified, through the gene expression by RT-PCR and the phenotypic profile of surface markers by FACS, that the increased susceptibility of murine AMs were due to a polarization of these cells into a M2 profile. To finish, we validated the relevance of thismodel through the analysis of the susceptibility of murine AMs deficient to NOS2, IFN-? and IL-4 as a proof of principle. We verified that the susceptibility of AMs is comparable to Vero cells and THP-1 cells, immortalized cell models described as highly susceptible to C. burnetii replication, and that AMs can be used to studies using cells derived from mice with the C57BL/6 background. In this way, our work characterized murine AMs as a relevant primary cell model highly susceptible to studies among the host-pathogen interactions in infections with C. burnetii phase II in vitro, and we also elucidate the mechanisms underlying this susceptibility

Pour survivre et se multiplier dans leurs cellules hôtes les bactéries intracellulaires ont élaboré divers mécanismes d'échappement à la réponse immunitaire. L’altération du trafic intracellulaire est une des stratégies utilisée par ces agents pathogènes. Ainsi Coxiella burnetii, bactérie intracellulaire stricte responsable chez l’homme de la fièvre Q, bloque la maturation du phagosome afin de résider et se multiplier dans un compartiment incapable de fusionner avec les lysosomes. Ce défaut de fusion est associé à la virulence bactérienne. Pour analyser le défaut de maturation du phagosome de C. burnetii, j’ai étudié le rôle du lipopolysaccharide (LPS) de C. burnetii dans le trafic intracellulaire de cette bactérie. J’ai montré que le LPS de C. burneti, unique par sa structure, ne permet pas l’activation de la MAPKinase p38α, entrainant un défaut dans le recrutement du complexe HOPS (homotypic fusion and vacuole protein sorting complex) nécessaire à la conversion phagosomale. J’ai en effet montré que le recrutement du complexe HOPS requiert la phosphorylation de la protéine Vps (vacuolar protein sorting) 41. La transfection de macrophages permettant la surexpression d’un activateur de p38 et l’utilisation de mutants phosphomimétiques de Vps41 ont montré une restauration de la conversion phagosomale. Il apparaît ainsi que la MAPK-p38α et son dialogue avec Vps41 jouent un rôle central dans la maturation du phagosome de C. burnetii en phagolysosome. L’utilisation de la structure atypique de son LPS permet ainsi à C. burnetii de se soustraire à la réponse protectrice de l'hôte. / To survive and replicate in their host, microbes have evolved several strategies to hijack the microbicidal properties of the immune cells. C. burnetii, the q fever agent, survive and replicate in macrophages through the alteration of the phago-lysosome biogenesis. To further analyze the nature of the defect phagosome maturation of C. burnetii, I studied the role of lipopolysaccharide (LPS) of C. burnetii in the intracellular trafficking of the bacteria. The LPS is unable to activate the p38α MAPKinase, which explains that the virulent bacteria are not directed to a degradative compartment . The lack of activation of the p38α MAPKinase , which involves a commitment TLR4 antagonist by LPS, has the effect of preventing the recruitment of the HOPS complex ( homotypic fusion and vacuole protein sorting complex) , a complex require for the phagosomal conversion. I have shown that the recruitment of HOPS requires phosphorylation of protein Vps (vacuolar protein sorting) 41. Transfection of macrophages by an activator of p38 and using phosphomimétiques mutants VPS41 showed restoration of phagosome maturation. It thus appears that the p38α MAPK and his dialogue with VPS41 play a central role in phagosome maturation of C. burnetii in the phagolysosome. Use of the unique structure of the LPS allows C. burnetii to evade the protective response of the host.

Les granulomes signent une réponse immune efficace dans de nombreuses maladies infectieuses. C’est le cas de la fièvre Q où ils sont présents dans la forme aiguë de la maladie spontanément résolutive mais où ils sont absents dans sa forme chronique. J’ai mis au point une méthode permettant d’étudier la formation in vitro des granulomes à partir de cellules mononucléées du sang périphérique incubées en présence de billes de Sepharose recouvertes d’extraits de C. burnetii, l’agent de la fièvre Q. Ces granulomes apparaissent en quelques jours avant de disparaître au bout d’une vingtaine de jours. Ils sont composés essentiellement de macrophages et de lymphocytes et, à un moindre degré, de cellules épithélioïdes, de cellules géantes multinucléées et de cellules dendritiques. La méthode d’obtention in vitro des granulomes a permis également d’étudier les premiers stades de leur formation grâce à la mise au point d’une technique en live imaging d’observation en lumière transmise des cellules. J’ai montré que les monocytes, les premières cellules à migrer vers les billes et à entièrement les recouvrir, initient le recrutement des lymphocytes. J’ai également montré que le défaut de formation des granulomes observés chez des patients atteints d’une fièvre Q chronique pourrait être lié à un défaut de migration de leurs monocytes circulants. / Granulomas indicate effective immune response in a large number of infectious diseases. The primo-infection by Coxiella burnetii, the agent of Q fever, is spontaneously resolutive and is characterized by the presence of granulomas, whereas granulomas are absent in the chronic form of the disease. I developed a new method to study in vitro the formation of granulomas using peripheral blood mononuclear cells incubated in the presence of Sepharose beads coated with C. burnetii extracts. Granulomas appeared in few days before disappearing at day 20. They were essentially composed of macrophages and lymphocytes and, in a lesser extent, of epithelioid cells, multinucleated giant cells and dendritic cells. The method to obtain in vitro granulomas allowed the study of the first events of granuloma formation in live imaging microscopy. Monocytes migrated toward Sepharose beads and entirely covered these beads, and initiated the recruitment of lymphocytes. Finally, mononuclear cells from patients with Q fever were unable to generate granulomas due to defective migration of monocytes. We hypothesize that defective migration of monocytes may be responsible for the defective formation of granulomas in Q fever.

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.

The zoonosis Q fever is caused by the obligate intracellular bacterium Coxiella burnetii. Besides the main transmission route via inhalation of contaminated aerosols, ticks are discussed as vectors since the first isolation of the pathogen from a Dermacentor andersonii tick. The rare detection of C. burnetii in ticks and the difficult differentiation of C. burnetii from Coxiella-like endosymbionts (CLEs) are questioning the relevance of ticks in the epidemiology of Q fever. In this review, literature databases were systematically searched for recent prevalence studies concerning C. burnetii in ticks in Europe and experimental studies evaluating the vector competence of tick species. A total of 72 prevalence studies were included and evaluated regarding DNA detection methods and collectionmethods, country, and tested tick species. Specimens ofmore than 25 different tick species were collected in 23 European countries. Overall, an average prevalence of 4.8% was determined. However, in half of the studies, no Coxiella-DNA was detected. In Southern European countries, a significantly higher prevalence was observed, possibly related to the abundance of different tick species here, namely Hyalomma spp. and Rhipicephalus spp. In comparison, a similar proportion of studies used ticks sampled by flagging and dragging or tick collection from animals, under 30% of the total tick samples derived from the latter. There was no significant difference in the various target genes used for the molecular test. In most of the studies, no distinction was made between C. burnetii and CLEs. The application of specific detection methods and the confirmation of positive results are crucial to determine the role of ticks in Q fever transmission. Only two studies were available, which assessed the vector competence of ticks for C. burnetii in the last 20 years, demonstrating the need for further research.