Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Francisella tularensis is a highly infective, intracellular bacterium. It is capable of infecting a wide range of mammals and causes the disease tularemia in humans. As a result of its high infectivity there have been a lot of efforts made to create a generally available vaccine against this pathogen. One potential vaccine candidate is the FSC043 strain, a spontaneous mutant that has acquired mutations making it attenuated for replication both in vitro and in the experimental mouse model. However, it was noted that it afforded protection against challenge with a highly virulent F. tularensis strain. The aim of this thesis has been to delineate the mechanisms of its attenuation to better understand F. tularensis pathogenesis and to obtain a better knowledge about the prerequisites of protective immunity against this potent pathogen. Microarray and whole-genome sequencing revealed four mutations in the attenuated FSC043 strain that were not present in the virulent SCHU S4 isolate. One of these mutations has been described earlier as it results in a fusion protein also found in other attenuated strains. Among the other differences, two mutations were identical nonsense mutations in a duplicated gene region known as the Francisella pathogenicity island (FPI). The affected gene, pdpC, is coding for PdpC (pathogenicity determinant protein C). We found that these mutations resulted in a truncated form of PdpC, and also that the downstream gene was severely downregulated due to these mutations. Further, our studies revealed that the intracellular phenotype of the FSC043 strain differed from other tested strains in that a small portion of the intracellular bacteria were able to escape the phagosome and multiply within the host, while the majority of intracellular bacteria stayed confined to the phagosome. We wanted to study the specific function of pdpC and therefore deleted both copies of it in the virulent SCHU S4strain as well as the Live Vaccine Strain, an empirically attenuated strain often used as a model for the virulent strains of F. tularensis. The resulting mutants showed an attenuated phenotype; no intracellular growth in murine cells, and no virulence in mice. When studying the intracellular localization of the LVS Δpdpc mutant, we found that it was uniformly located adjacent to phagosomal membrane-like structures but that the membrane was markedly disrupted. Further, this mutant induced an MOI-dependent cytotoxicity, measured by LDH release, and also the release of IL-1β, an inflammatory cytokine not induced by phagosomally contained mutants. Studies on markers for host cell death revealed that the LVS ΔpdpC mutant induced mitochondrial instability, phosphatidylserine (PS) presentation, and TUNEL-specific DNA fragmentation in infected cells, rather similar to the wild-type strain, despite its lack of replication. This study reveals that the pdpC gene is an important gene required for F. tularensis virulence. We also show that non-replicating intracellular bacteria can induce host cell death, hypothesizing that release of bacterial components in the host cell cytosol is required for this induction. The FSC043 mutant showed a unique phenotype where a small subset of bacteria was able to escape the phagosome and replicate in the host cell. This was also seen in the pdpC deletion mutant of SCHU S4, but not with the LVS ΔpdpC. However, regardless of genetic background, the ΔpdpC mutant had an effect on phagosomal escape; either by affecting the phagosomal membranes in a unique way or by allowing phagosomal escape of a small proportion of the bacteria.

Blood was obtained from 5 adult horses and 16 juvenile horses (foals) at the time of birth and subsequently at 2-, 4-, and 8-weeks of age. Neutrophils from adult horses were purified and incubated for 2 h and 4 h with media, avirulent R. equi, virulent R. equi, or recombinant-human granulocyte-macrophage colony stimulating factor (rhGM-CSF). Neutrophils from foals were purified and incubated for 2 h and 4 h with media or virulent R. equi. Total RNA was extracted from both adult and foal neutrophils immediately after purification to measure baseline expression levels (0 h), and immediately after each of the prescribed incubation times. For each sample, 1 µg of total RNA was reverse-transcribed and analyzed for differential gene expression using real-time PCR. After 2 h and 4 h incubation with virulent or avirulent R. equi, neutrophils from adult horses expressed significantly (P< 0.05) greater TNFα, IL-12p40, IL-6, IL-8, and IL-23p19 mRNA relative to expression by unstimulated neutrophils, but not IFNγ or IL-12p35 mRNA. Furthermore, virulent R. equi induced significantly greater IL-23p19 mRNA expression than avirulent R. equi. Stimulation with rhGM-CSF of adult equine neutrophils failed to induce significant changes in cytokine expression. In foal neutrophils, stimulation with virulent R. equi induced significantly greater expression of IFNγ, TNFα, IL-6, IL-8, IL-12p40, and IL-12p35 mRNA relative to expression by unstimulated neutrophils. Furthermore, there were significant effects of age on expression of IL-6, IL-8 and IL-12p40 mRNA. Neutrophil mRNA expression of IL-6 and IL-8 in newborn foals was significantly greater than expression at 2-, 4-, and 8-weeks of age. There was no significant difference between unstimulated and R. equi-stimulated neutrophils from newborn and 2-week-old foals in expression of IL-12p40; however, expression of IL-12p40 by R. equi-stimulated neutrophils from 4- and 8-week-old foals was significantly greater than expression by unstimulated neutrophils. These results demonstrate that R. equi-stimulated neutrophils are a source of many pro-inflammatory cytokines, and that the magnitude of this expression with respect to IL-6, IL-8, and IL-12p40 mRNA expression was influenced by age. Collectively, the data presented indicate a non-phagocytic role for neutrophils that may influence the type of adaptive immune response to R. equi.

Macromolecular complexes of anionic-nonionic block copolymers and cationic antibiotic aminoglycosides have been formed by electrostatic condensation. Amphiphilicity of the complexes was introduced into the shells by incorporating a hydrophobic poly(propylene oxide) segment into the block copolymer. The resulting particles have an average hydrodynamic diameter of ~ 200 nm and contain up to 30-40 % of the drug payload. In vitro efficacies of such nanostructures in reduction of intracellular pathogens like Salmonella, Listeria, and Brucella were demonstrated. Current effort focuses on translation of this nano-drug delivery concept to in vivo model of intracellular infectious diseases. Atom transfer radical polymerization (ATRP) was utilized to prepare well-defined polymeric dispersion stabilizers that readily adsorb onto metal oxide surfaces. Two unimolecular bis(phosphonate) ATRP initiators were designed and prepared in good yield. These special initiators were successfully used to initiate polymerization of poly(N-isopropylacrylamide) (PNIPAM) in a controlled manner yielding PNIPAM with a bis(phosphonate) moiety at one terminus. The polymers readily adsorbed onto magnetite nanoparticle surfaces, thus creating thermosensitive magnetic nanostructures that form nanosized clusters upon heating above the lower critical solution temperature of PNIPAM. It is envisioned that modularity of this approach, relying on the applicability of ATRP to polymerize a vast array of monomers, could be used to prepare a library of polymeric shells for magnetic iron oxide nanoparticles. Medical intervention in drug delivery that includes detectability of drug carriers is greatly desirable. A real-time assessment of disease prognosis could be highly beneficial for developing personalized treatment strategies. As an example of this conceptual innovation, block ionomer functionalized magnetite complexes were synthesized and investigated as carriers for delivery of aminoglycosides into phagocytic cells for treatment of intracellular bacterial infections. The ionic block of copolymer contains multiple carboxylates for binding onto the iron oxide surface. The remaining unbound carboxylate anions were used to complex with cationic gentamicin in nanoshells of these complexes. The iron oxide particle core provides an imaging modality and serves as a pseudo-crosslinking site to enhance stabilities of the polyelectrolyte complexes, thus preventing them from disintegrating in the physiological environment. Currently, these hybrid complexes are being investigated in possible pharmaceutical formulations to eradicate intracellular pathogens in animal models. / Ph. D.

Une partie de ce travail de thèse a consisté à appliquer les méthodes de FISH pour l’étude de trois bactéries pathogènes intracellulaires. La viabilité de Bartonella henselae a été évaluée à partir de ganglions de patients atteints de la maladie des griffes du chat (CSD). Le faible taux d’ARN détecté par biologie moléculaire, la stérilité des cultures, l'absence de détection par analyses histologiques et FISH confirment que B. henselae n'est pas ou rarement viable dans les ganglions de patients atteints de CSD. Tropheryma whipplei, l’agent de la maladie de Whipple, a été identifié et localisé par FISH, dans les macrophages d’un ganglion et d’une biopsie pulmonaire, confirmant le diagnostic infectieux. Deux méthodes de FISH ont été testées pour détecter Coxiella burnetii dans des cas d’endocardites et d’infections vasculaires en utilisant des sondes oligonucléotidiques et des sondes PNA. Les résultats ont confirmé une meilleure efficacité des sondes PNA et démontré que les techniques de FISH sont plus sensibles que l’immunohistochimie pour le diagnostic des endocardites et des infections vasculaires à C. burnetii. Nous avons également évalué les stratégies moléculaires mises en place pour le diagnostic syndromique. Bien que la PCR conventionnelle à large spectre permette l'identification de micro-organismes fastidieux et anaérobies, la PCR spécifique en temps réel révèle une supériorité significative dans le diagnostic syndromique. En conclusion, ce travail a permis de démontrer l’efficacité et l’applicabilité de la FISH pour la détection bactérienne. Cette méthode peut être utilisée comme un outil complémentaire afin d'améliorer le diagnostic de microbiologie clinique. / We applied FISH methods to the study of three intracellular pathogenic bacteria. The viability of Bartonella henselae was evaluated in a large series of lymph nodes from patients with cat scratch disease (CSD). The results obtained, associated with sterile cultures and negative histological analyzes and FISH, as well as the low level of RNA detected by molecular biology, provide evidence that B. henselae are not or are rarely viable in the lymph nodes of patients with CSD. Tropheryma whipplei has been identified by FISH in macrophages from one lymph node and for the first time in a pulmonary biopsy, confirming the diagnosis of infection. Two methods of FISH have been tested to detect Coxiella burnetii in cases of endocarditis and vascular infections using oligonucleotide and PNA probes. The results attested to the greater efficiency of PNA probes, and demonstrated that FISH were applicable for the diagnosis of C. burnetii endocarditis. We also evaluated the molecular strategies used for syndrome-driven diagnosis of infectious diseases. Although conventional broad-spectrum PCR allows for the identification of fastidious and anaerobic microorganisms, real-time specific PCR reveals a significant superiority in syndrome-driven diagnosis. The addition of specific PCRs in real time PCR would improve our molecular strategies, for example, in the case of the detection of Staphylococcus aureus for the diagnosis of lymphadenopathy. In conclusion, this work demonstrates the effectiveness and applicability of FISH for the identification of intracellular bacteria. This method can be used as an important complementary tool to the improvement of clinical microbiological diagnosis.

AU COURS DE LA THESE, L'ETUDE DU TRANSCRIPTOME DE SOUCHES GARDEL ET SENEGAL VIRULENTES ET ATTENUEES D'E. RUMINANTIUMA ETE REALISEE. UNE ANALYSE DU TRANSCRIPTOME A DIFFERENTS STADES DE DEVELOPPEMENT, A D'ABORD ETE EFFECTUEE POUR LA SOUCHE GARDEL VIRULENTE. AU STADE CORPS RETICULE (FORME INTRACELLULAIRE NON INFECTIEUSE), UNE SUREXPRESSION DES GENES CODANT POUR DES PROTEINES IMPLIQUEES DANS LE METABOLISME, LE TRANSPORT ET L'ECHANGE DE NUTRIMENTS ET DANS LA RESISTANCE AU STRESS OXYDATIF ETAIT OBSERVEE. IL SEMBLERAIT QUEE. RUMINANTIUMMETTE EN PLACE UN PANEL DE MECANISMES POUR SA SURVIE ET SON DEVELOPPEMENT A L'INTERIEUR DE LA CELLULE HOTE. AU STADE CORPS ELEMENTAIRE (FORME EXTRACELLULAlRE INFECTIEUSE), LE GENE DKSA CODANT POUR UN FACTEUR DE TRANSCRIPTION ETAIT SUREXPRIME. CE GENE A ETE MONTRE COMME ETANT IMPLIQUE DANS LA REGULATION DE FACTEURS DE VIRULENCE. IL SEMBLERAIT . DONC, QU'AU STADE CORPS ELEMENTAIRE, IL Y AIT UNE INDUCTION DE MECANISMES DE VIRULENCE. LA COMPARAISON DE L'EXPRESSION DES GENES AU STADE CORPS ELEMENTAIRE ENTRE SOUCHES VIRULENTES ET ATTENUEES A AUSSI ETE EFFECTUEE. NOS RESULTATS ONT MONTRE UNE MODIFICATION IMPORTANTE DE LA MEMBRANE POUR LES SOUCHES VIRULENTES ET ATTENUEES. POUR LES SOUCHES ATTENUEES, IL A ETE MONTRE UNE SUREXPRESSION DES GENES IMPLIQUES DANS LA BIOGENESE MEMBRANAlRE ET UNE SOUS-EXPRESSION·DES PROTEINES DE LA FAMILLE MULTIGENIQUE MAP. CES RESULTATS SUGGERENT QUE LES PROTEINES MAP JOUENT UN ROLE DE LEURRE VIS-A-VIS DE LA REPONSE IMMUNITAIRE PROTECTRICE. DES PROTEINES MEMBRANAlRES HYPOTHETIQUES SONT SUREXPRIMEES A LA FOIS CHEZ LES SOUCHES VIRULENTES ET ATTENUEES. CERTAINES D'ENTRE ELLES SUREXPRIMEES CHEZ LES SOUCHES ATTENUEES SEMBLENT ETRE DE BONS CANDIDATS VACCINAUX ET DEVRAIENT ETRE ETUDIEES / Transcriptomic study of gardel and senegal both virulent and attenuated e. ruminantium strains was conducted during my phd. an analysis of transcriptome at different stages of development has been first conducted for virulent gardel strain. at reticulate body stage (intracellular form non-infectious), over-expression of genes coding for proteins involved in metabolism, transport and exchange of nutrients and resistance to oxidative stress was observed. at this stage of development, e. ruminantium seems to activate mechanisms for its survival and development within the host cell. at elementary body stage, dksa the gene encoding for a transcription factor was over-expressed. this gene has been shown to be involved in the regulation of virulence factors. it seems, therefore, at the elementary body stage, e. ruminantium induces its virulence factors. secondly, we compare the transcriptome of elementary body between virulent and attenuated strains. our results showed an important membrane modification of attenuated and virulent strains. for attenuated strains, we observed an over-expression of genes involved in membrane biogenesis and a diminution of expression of map multigenic family. it seems that map proteins subvert the protective immune response. hypothetical membrane proteins are over-expressed in both virulent and attenuated strains. some over-expressed proteins in attenuated strains seem to be good vaccine candidates and willstudied.

Therapeutically restricted sites present a formidable barrier in medicine. Herein, chemical strategies to overcome two restricted sites, HIV reservoirs and intracellular bacteria, will be discussed. First, cellular and anatomical HIV reservoirs, such as those in the brain, limit HIV eradication using currently known therapeutic regimes. HIV therapies are unable to localize in the brain, in part, due to high expression of efflux transporters, such as P-glycoprotein (P-gp), at the BBB, because many of these therapies are P-gp substrates. In an effort to overcome therapeutically restricted HIV sanctuaries, a dimerized combination HIV therapy was designed to act two-fold. First, the dimeracts as a P-gp inhibitor allowing therapeutic access to restricted sites. Second, the dimeractsas a prodrug, which once in the reducing environment of the cell, may release monomeric HIV therapies. The dual conjugate, Abacavir-S2-Darunavir, was shown to potently inhibit P-gp across two separate cell lines, was able to regenerate the component monomers in a reducing environment and contained modest anti-HIV activity.<div><br><div>Further, mammalian cells create sanctuary sites for bacteria to grow and proliferate, because many common antibiotic therapies are unable to cross the mammalian cell membrane. Therefore, these pathogens are able to proliferate without therapeutic constraint. Here, a chemical strategy was developed to deliver a dual antibiotic therapy inside mammalian cells in an effort to clear these intracellular pathogens. First, a new synthetic strategy was developed for facile synthesis of dual conjugates, composedof an aminoglycoside and a cell penetrating peptide (CPP) linked with a reversible disulfide tether, using kanamycin and the known CPP Arg8as a model system. Next, this synthetic methodology was expanded for use with theaminoglycoside tobramycin and theknown broad-spectrum antibiotic and cell penetrating peptide, P14LRR, once again linked via the reversible disulfide tether (TobP14). Two distinct isomers of TobP14 were synthesized, isolated, and fully characterized by 2D NMR. The TobP14 isomers were shown to be an effective antibiotic across various Gram positive and negative pathogens such as MRSA, S. epidermidis, P. aeruginosa, and A. baumannii. Further, the isomers effectively releasedthe monomeric therapies (tobramycin and P14-SH) in a reducing environment and werenontoxic to mammalian cells up to 16 μM. Finally, the dual conjugate isomers significantly reduce two different strains of intracellular A. baumanniiwithin macrophages.<br><div><br></div><div><br></div></div></div>

The intracellular bacteria Wolbachia infect up to 40% of all insect species, including the vectors of prevalent infectious diseases such as Dengue and malaria. Even though Wolbachia infections are the largest pandemic on this planet, the cellular and molecular mechanisms for bacterial spreading in nature are still unknown. Wolbachia are mainly vertically transmitted through the egg cytoplasm, however there is also evidence of extensive horizontal transmission. We have found that Wolbachia target the stem cell niches in the Drosophila ovary to enhance germline colonization and subsequent vertical transmission. This tropism is pervasive across the Drosophila genus, with the pattern of targeting being evolutionarily conserved. Phylogenetic analyses, confirmed by hybrid introgression and transinfection experiments, demonstrate that bacterial factors are the major determinants of differential patterns of niche tropism. Furthermore, bacterial load is increased in germline cells passing through infected niches, supporting previous findings suggesting a contribution of Wolbachia from stem cell niches towards vertical transmission. If niche tropism is important for Wolbachia transmission through the germline, evolutionary theory predicts that there should be no selective pressure to maintain niche tropism in males. Indeed, we have found that tropism to the stem cell niche in the testis, known as the hub, is not evolutionarily conserved. Towards identifying the cellular and molecular mechanisms of stem cell niche tropism, we investigated hub targeting of closely related Wolbachia strains (wMel-like strains: wMel, wMel2, and wMel3; wMelCS-like strains: wMelCS, wMelCS2, and wMelPop). wMel-like and wMelCS-like Wolbachia strains differ in their frequencies and densities of hub infection. The targeting differences of these strains of Wolbachia indicate that this phenotype is rapidly evolving, as they shared a common ancestor only 8,000 years ago. With the plethora of tools available in D. melanogaster, a candidate gene approach was used to target host proteins enriched in the stem cell niche in the testis for RNAi mediated gene knockdown in the hub. We have identified Drosophila stem cell related signaling pathways that promote Wolbachia accumulation. Unraveling the cellular and molecular bases of tissue tropism is fundamental to understanding Wolbachia-host interactions. / 2017-01-01T00:00:00Z

Les bactéries appartenant au complexe Burkholderia cepacia (Bcc) sont des pathogènes opportunistes intracellulaires qui causent des infections pulmonaires chez les patients atteints de mucoviscidose, aggravant leur pronostic clinique. Ces infections pulmonaires sont caractérisées par des périodes chroniques avec des exacerbations intermittentes détériorant la fonction pulmonaire, et pouvant causer des nécroses broncho-pulmonaires et septicémies fatales reconnues sous le nom du “Syndrome Cepacia”. Ces bactéries intrinsèquement multi-résistantes aux antibiotiques sont aussi responsables de sérieuses infections émergentes dans des contextes hors mucoviscidose, à la fois dans des conditions intra et extra-hospitalières. Burkholderia cenocepacia, l’une des espèces les plus répandues et isolées chez les patients, est capable d’échapper à la dégradation par les macrophages de l’hôte en bloquant la maturation des (auto)phagosomes. Nous avons récemment démontré que les macrophages servent de niche essentielle pour la réplication intracellulaire de B. cenocepacia K56-2, et sont nécessaires pour le développement d’une réponse pro-inflammatoire aiguë et fatale dans des larves de poisson zèbre. Cette étude exploite d’autant plus le modèle du poisson zèbre pour mieux comprendre quels sont les facteurs bactériens et de l’hôte qui sont impliqués dans la différence entre infection aiguë et persistante, et dans la transition entre ces deux phases infectieuses.ShvR est un régulateur transcriptionnel appartenant aux LTTRs (“LysR-Type Transcriptional Regulators”) chez B. cenocepacia K56-2. Il a été démontré dans le modèle d’infection pulmonaire chez le rat, que ShvR a un rôle important dans l’induction de la réponse pro-inflammatoire, mais pas dans les infections persistantes. Pour cette étude nous utilisons une approche bioinformatique, le modèle du poisson zèbre et des études transcriptomiques afin d’obtenir plus d’informations sur le rôle de ShvR dans la virulence et dans la transition entre infection persistante et réponses pro-inflammatoires. Nos données bioinformatiques suggèrent que le gène shvR s’est adapté par évolution divergente, et a été perdu dans une sous-classe du Bcc. Grâce au modèle du poisson zèbre, nous avons démontré que ShvR n’est pas essentiel pour les stades intra-macrophagiques, mais qu’il est requis pour la dissémination de B. cenocepacia K56-2 des macrophages et pour le développement d’une réponse pro-inflammatoire fatale. Le profile persistant de l’infection a été confirmé par l’analyse du transcriptome de l’hôte, donnant plus d’informations sur les différentes réponses de l’hôte envers les infections par des mutants comparées à la souche sauvage. Le travail de cette thèse a contribué non seulement à une meilleure compréhension du rôle de ShvR et aux gènes cibles régulés par ce dernier qui joue un rôle important dans les infections aiguës, mais également à établir de nouvelles pistes pour développer de nouvelles stratégies thérapeutiques luttant contre les infections par les bactéries appartenant au complexe Bcc. / Bacteria belonging to the Burkholderia cepacia complex (Bcc) are opportunistic pathogens with an intracellular life style. Pulmonary infections with these bacteria significantly worsen clinical outcome for cystic fibrosis (CF) patients. Chronic infections with recurrent acute exacerbations deteriorate lung function with sometimes fatal necrotizing pneumonia and septicaemia (Cepacia Syndrome). These intrinsically multi resistant bacteria are also emerging as the culprit of serious infections in non-CF settings, both in- and outside the hospital. B. cenocepacia, one of the more prevalent species in the complex, is able to avoid degradation by host macrophages by arresting (auto)phagosome maturation. We have recently shown that macrophages provide a critical site for intracellular replication of B. cenocepacia K56-2 and development of acute fatal pro-inflammatory infection in zebrafish larvae. This study further explores the zebrafish infection model to better understand bacterial and host factors involved in the difference between persistent and acute infection, and the transition between these stages.ShvR, a LysR-type transcriptional regulator of B. cenocepacia K56-2, has been shown to play an important role in the induction of pro-inflammatory responses in a rat lung infection model, but not in persistent infection. We used bioinformatics, the zebrafish infection model, and host transcriptome profiling to gain more insight into the role of ShvR in virulence, and in transition between persistent and pro-inflammatory responses. Our bioinformatics study suggests that shvR has adapted by divergent evolution, and has been lost in a subclade of the Bcc. Using the zebrafish embryo model, we demonstrate that ShvR is not important for intramacrophage stages, but is required for dissemination of B. cenocepacia K56-2 from infected macrophages and the development of pro-inflammatory fatal disease. The persistent character of the infection was confirmed by host transcriptomic analysis, giving insight into the differential host response towards the mutant compared to wildtype infection. This thesis contributes to a better understanding of the role of ShvR and its possible target genes that play an important role in acute infection and to future perspectives of development of new targets for the treatment of Bcc infections.