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.

Les infections causées par Staphylococcus aureus sont associées à un fort taux de mortalité au sein de la société. En plus d'être responsable de diverses infections, cette bactérie possède une panoplie de résistances aux antibiotiques ce qui rend son traitement beaucoup plus difficile. La vaccination semble être la voie la plus prometteuse compte tenu de l'inefficacité des antibiotiques. Bien que cette bactérie cause beaucoup de dommages, il est difficile d'enrayer toute source de S. aureus puisque celle-ci est un commensal de l'humain et se retrouve au niveau du nez et sur la peau de porteurs sains. L'étude de l'efficacité d'un vaccin suite à une infection septicémique avec différentes souches de S. aureus chez la souris était le principal objectif du présent projet. Il s'agissait donc de comparer la protection immunitaire engendrée par le vaccin face à ces souches. Les souches SHY97-3906 (isolat de mammite), MRSA256c (isolat nasal chez l'humain) et Newman (souche contrôle séquencée, isolée au départ d'une ostéomyélite) ont été utilisées. Le vaccin que nous avons employé est composé de quatre protéines différentes hautement conservées chez les différentes souches de S. aureus soit; IsdB (iron-regulated surface determinant ), HarA/IsdH (haptoglobin receptor A ), CIfA (clumping factor A ) et GapC/B (glycéraldéhyde-3-phosphate déshydrogénase). De plus, deux adjuvants ont été utilisés, le PCEP (polyphosphazene ) et le pGM-CSF (granulocyte-macrophage colony-stimulating factor plasmidique). Ces différents éléments de notre vaccin ont été choisis suite à des résultats préliminaires obtenus au laboratoire par Marie Rivest. Ce vaccin a donc été testé chez la souris (CD-1) face à une infection septicémique. Suite à des résultats non concluants, l'objectif de l'étude s'est plutôt redirigé vers l'investigation de l'échec du vaccin face à la septicémie. Puisque le vaccin ne semblait pas pouvoir contenir l'infection, il était important de vérifier l'efficacité des anticorps à lier la bactérie. Une grande variabilité a été obtenue ce qui a poussé l'analyse plus loin, au niveau de l'expression des gènes cibles. Des PCR réguliers et quantitatifs ont été réalisés sur les différentes souches afin de déterminer les différences d'expression. De plus, lors de mise au point du modèle de septicémie des différences notables de virulence in vivo avaient été observées entre les souches de S. aureus . Un lien a donc pu être fait entre l'habileté des anticorps à lier les souches, l'expression des protéines cibles et la virulence des souches afin d'expliquer en partie, l'échec de notre vaccin face à S. aureus . Comme il a été démontré dans la littérature que les individus portant S. aureus au niveau du nez étaient plus enclins à développer des infections, une mise au point du modèle nasal a été effectuée. Cependant, aucun résultat significatif au niveau de la protection n'a été obtenu.

Clostridium difficile est une bactérie Gram positive anaérobique responsable de plusieurs perturbations intestinales suite à la prise d’antibiotiques et est également reconnue comme une cause majeure de la colite pseudomembraneuse. La pathogenèse des infections à C. difficile est principalement associée aux toxines A et B, qui ont été largement étudiées dans les dernières années. Cependant, plusieurs autres aspects de la virulence de ce pathogène sont encore bien mal compris. Un bon nombre de protéines font partie de l’arsenal de facteurs de virulence qui jouent un rôle au niveau de l’adhésion de la bactérie ou qui dégradent certaines composantes de la paroi intestinale afin de permettre à la bactérie de coloniser son hôte. La régulation de l’expression de telles protéines nous permettrait d’en connaître davantage sur la colonisation de l’hôte par C. difficile. Chez plusieurs espèces bactériennes, le 3’5’ diguanosine monophosphate cyclique (c-di-GMP) est un second messager important qui régule plusieurs phénotypes. La formation de biofilm ainsi que la régulation de la transition de cellules motiles à un mode de persistance sont parmi les fonctions connues du c-di-GMP. Toutefois, la diversité des processus physiologiques bactériens régulés par le c-di-GMP reste encore pour la plupart peu expliquée chez les bactéries à Gram positif. Chez certaines bactéries, le c-di-GMP est impliqué dans le contrôle de la régulation de l’expression de gènes, via la liaison à des récepteurs à ARN, les riborégulateurs. Les riborégulateurs sont des séquences d’ARN non codants localisés dans la partie 5’UTR d’un ARNm et régulant l’expression de gènes par la liaison d’un ligand à son aptamère comme le c-di-GMP. Cette étude s’intéresse à la régulation et à la fonction des gènes CD2831 et CD2830 qui sont regroupés en un seul locus. L’expression de ces gènes est contrôlée par les niveaux de c-di-GMP intracellulaire via deux riborégulateurs en amont de ces gènes. Chez C. difficile, deux classes de riborégulateurs à c-di-GMP, de fonction similaire, mais qui diffèrent structurellement, ont été rapportées; c-di-GMP-I et c-di-GMP-II. L’expression relative du gène CD2830 en aval du riborégulateur à c-di-GMP de type I diminue de 23 à 29 fois lorsque le niveau de c-di-GMP intracellulaire augmente, tandis que pour les mêmes conditions, le gène CD2831 en aval du riborégulateur à c-di-GMP II était de 72 à 96 fois plus exprimé. Des essais β-galactosidase avec des fusions des riborégulateurs et lacZ, ont démontrés que l’expression des gènes CD2830 et CD2831 semble être contrôlée de façon opposée par leurs riborégulateurs transcriptionnels respectifs, selon les concentrations de c-di-GMP intracellulaire. La protéine CD2830 a été décrite comme une métalloprotéase zinc-dépendante ayant une activité sur la fibronectine, le fibrinogène de plasma humain et la protéine CD2831, qui code pour une adhésine putative. La fonction de cette dernière protéine reste encore mal connue. Cette étude n’a pas permis de démontrer que les protéines CD2830 et CD2831 jouent un rôle dans l’agrégation ou dans l’adhésion à la fibronectine et au fibrinogène lorsque la concentration de c-di-GMP intracellulaire varie. Cette étude suggère que chez C. difficile, la zinc-métalloprotéase CD2830 est impliquée dans un mécanisme de clivage de protéines d’adhésion, comme décrit chez plusieurs pathogènes, afin de contrôler l’adhésion cellulaire lors de processus biologique important. Ce mécanisme serait régulé par le c-di-GMP.

Streptococcus pneumoniae is a commensal inhabitant of the human nasopharynx that can sometimes become pathogenic and cause diseases such as pneumonia, otitis media and meningitis. Carbohydrate metabolism is a critical component of S. pneumoniae virulence. Among the myriad of carbohydrate-specific pathways involved in the host-pneumococcus interaction, the N-glycan foraging pathway stands out because of its direct implication in numerous aspects of virulence such as fitness, adhesion/invasion and impairment of the host immune response. Much of the literature has been focussed on the importance of step-wise depolymerisation of N-glycans by the enzymes NanA, BgaA and StrH. However, the importance of the liberation of N-glycans from host glycoconjuguates and their intake by the bacterium has yet to be examined. We have identified a Carbohydrate Processing Locus (CPL) that is highly conserved throughout a large number of Firmicutes and whose individual components appear widespread in bacteria that we hypothesize is active on host N-glycans. This locus encodes for two putative α-mannosidases GH92 and GH38, a characterised α-mannosidase GH125, a putative β-hexosaminidase GH20C, a putative α-fucosidase GH29 and a ROK (Repressor, Open reading frame, Kinase) protein. The genomic context of CPL orthologues suggests that an endo-β-N-acetylglucosaminidase (EndoD) and an ABC transporter (ABCN-glycan) are functionally associated with this locus. Based on our bioinformatic analyses and known functions of these proteins we hypothesize that the CPL encodes a concerted pathway responsible for the liberation, transport, and processing of N-glycans. The objective of this research is to characterize the putative components of this pathway and assess their implication in virulence. Specific focus on ABCN-glycan demonstrated its specificity for a range of N-glycans liberated by EndoD, shedding light on a novel import system for branched N-glycans. Furthermore, we provided evidence that GH92 is an α-1,2-mannosidase that likely removes the terminal mannose residues found on high-mannose N-glycans. EndoD and GH92 are shown to participate in virulence in mice; however, their role in virulence has yet to be determined. This work will significantly advance the construction and validation of a model of N-glycan processing by S. pneumoniae. As the components of this model pathway are conserved amongst a wide variety of bacteria, this work is of fundamental relevance to understanding how microbes from various environments degrade and metabolize N-glycans. / Graduate

No description available.

A HEp-2 cell culture model was used to investigate the invasive properties of Campylobacter species. Two of twenty-five Campylobacter isolates did not invade HEp-2 cells, and one of these isolates did not adhere to the epithelial cells. Penetration of HEp-2 epithelial cells by C. jejuni was significantly (P < 0.05) inhibited with C. jejuni lysates and a MAb (1B4) in competitive inhibition studies. Immunogold electron microscopic studies revealed that the 1B4 MAb bound to the flagella and cell surface of low passage (invasive) C. jejuni M 96, whereas only the flagella of high passage (non-invasive) C. jejuni were labelled. Western blot analysis revealed that the 1B4 MAb identified an epitope on antigens ranging in size from 66 to 44 kDa in invasive and non-invasive organisms. Antigens were also recognized in lysates prepared only from invasive strains from 42 to 38 kDa. Sodium meta-periodate chemical treatment of C. jejuni lysates significantly (P < 0.05) affected its inhibitory capacity. Additionally, proteinase K and sodium meta-periodate treatment of lysates changed the mobility of antigens recognized by the 1B4 MAb. This suggests that the antigens required for epithelial cell penetration by C. jejuni may be glycoprotein in nature and that the functional binding site is dependent upon an intact carbohydrate moiety. Co-infection of HEp-2 epithelial cells with coxsackievirus B3, echovirus 7, polio virus (LSc type 1), porcine enterovirus and Campylobacter isolates was performed to determine if a synergistic effect could be obtained. The invasiveness of C. jejuni was significantly increased for HEp-2 cells pre-infected with echovirus 7, coxsackievirus B3, and UV-inactivated (non-infectious) coxsackievirus B3 particles. Polio and porcine enterovirus had no effect on C. jejuni adherence and invasiveness. C. hyointestinalis and C. mucosalis, two non-invasive isolates, did not invade virus-infected HEp-2 cells. The increase of invasiveness of C. jejuni appears to be the result of specific interactions between the virus and the HEp-2 cell membrane. The data suggest that the invasiveness of Campylobacter is dependent upon the inherent properties of the organism. Virus-induced cell alterations can potentiate the invasiveness of virulent Campylobacter but are not sufficient to allow internalization by non-invasive bacteria.

Biological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China.

<p>While the many completely sequenced genomes of bacterial pathogens contain all the determinants of the host-pathogen interaction, and also every possible drug target and recombinant vaccine candidate, computational tools for selecting suitable candidates for further experimental analyses are limited to date. The overall objective of my PhD project was to attempt to design reusable systems that employ the two most important features of bacterial evolution, horizontal gene transfer and adaptive mutation, for the identification of potentially novel virulence-associated factors and possible drug targets. In this dissertation, I report the development of two novel technologies that uncover novel virulence-associated factors and mechanisms employed by bacterial pathogens to effectively inhabit the host niche. More importantly, I illustrate that these technologies may present a reliable starting point for the development of screens for novel drug targets and vaccine candidates, significantly reducing the time for the development of novel therapeutic strategies. Our initial analyses of proteins predicted from the preliminary genomic sequences released by the Sanger Center indicated that a significant number appeared to be more similar to eukaryotic proteins than to their bacterial orthologs. In order determine whether acquisition of genetic material from eukaryotes has played a role in the evolution of pathogenic bacteria, we developed a system that detects genes in a bacterial genome that have been acquired by interkingdom horizontal gene transfer.. Initially, 19 eukaryotic genes were identified in the genome of Mycobacterium tuberculosis of which 2 were later found in the genome of Pseudomonas aeruginosa, along with two novel eukaryotic genes.</p> <p>Surprisingly, six of the M. tuberculosis genes and all four eukaryotic genes in P. aeruginosa may be involved in modulating the host immune response through altering the steroid balance and the production of pro-inflammatory lipids. We also compared the genome of the H37Rv M. tuberculosis strain to that of the CDC- 1551 strain that was sequenced by TIGR and found that the organisms were virtually identical with respect to their gene content, and hypothesized that the differences in virulence may be due to evolved differences in shared genes, rather than the absence/presence of unique genes. Using this observation as rationale, we developed a system that compares the orthologous gene complements of two strains of a bacterial species and mines for genes that have undergone adaptive evolution as a means to identify possibly novel virulence &ndash / associated genes. By applying this system to the genome sequences of two strains of Helicobacter pylori and Neisseria meningitidis, we identified 41 and 44 genes that are under positive selection in these organisms, respectively. As approximately 50% of the genes encode known or potential virulence factors, the remaining genes may also be implicated in virulence or pathoadaptation. Furthermore, 21 H. pylori genes, none of which are classic virulence factors or associated with a pathogenicity island, were tested for a role in colonization by gene knockout experiments. Of these, 61% were found to be either essential, or involved in effective stomach colonization in a mouse infection model. A significant amount of strong circumstantial and empirical evidence is thus presented that finding genes under positive selection is a reliable method of identifying novel virulence-associated genes and promising leads for drug targets.</p>

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, we decided to test the ability of the DHOase mutant strain to produce other virulence factors as well. Here we report that a block in the conversion of carbamoyl aspartate (CAA) to dihydroorotate significantly impairs the ability of P. aeruginosa to affect virulence. We believe that the accumulation of CAA in the cell is the root cause of this observed defect. This research demonstrates a potential role for pyrimidine intermediates in the virulence response of P. aeruginosa and may lead to novel targets for chemotherapy against P. aeruginosa infections.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Juergen A. Richt / Rift Valley fever virus (RVFV) is a vector-borne zoonotic pathogen endemic to sub-Saharan Africa and the Arabian Peninsula that causes severe disease in ruminants and humans. RVFV is a significant threat to US livestock and public health due to a lack of licensed, efficacious vaccines and its ability to become established in non-endemic areas. Subunit vaccine candidates based on RVFV N- and C-terminal glycoproteins (Gn and Gc) are a viable option for use in ruminants due to their ease of production, safety, and ability to induce immune responses that offer differentiation between infected and vaccinated animals (DIVA). Importantly, subunit Gn+Gc vaccine candidates have demonstrated efficacy in sheep. However, despite the efficacy of a dual glycoprotein vaccine, no studies have directly compared protective efficacies of the individual glycoproteins. Furthermore, although RVFV demonstrates 2.1% maximum pairwise amino acid strain divergence within Gn/Gc ectodomains, it remains unclear how this may affect cross-protective vaccine efficacy. In this study, we used a BALB/c mouse model to determine the median lethal dose (LD₅₀) of 3 wildtype RVFV strains and used this information to standardize challenge doses in subsequent vaccine efficacy studies using baculovirus-expressed Gn/Gc antigens derived from RVFV strain Zagazig Hostpital 1977 (ZH548). Strains Kenya 2006 (Ken06) and Saudi Arabia 2001 (SA01) demonstrated equally high virulence (LD₅₀= 7.9pfu), while recombinant strain South Africa 1951 (rSA51) was less virulent (LD₅₀=150pfu). Following prime-boost vaccination, 100% (10/10) of the Gn+Gc vaccinated mice survived challenge with x1000 LD₅₀ Ken06 and SA01, while only 50% (5/10) of Gn+Gc vaccinated mice survived challenge with rSA51. Additionally, 90% (9/10) of Gn-only vaccinated and 40% (4/10) of Gc-only vaccinated mice survived challenge with Ken06. These data suggest that a Gn-only subunit vaccine is an efficacious alternative to dual glycoprotein vaccine candidates and that our ZH548-derived Gn+Gc vaccine has the potential to cross-protect against divergent RVFV strains. Results from this study can be used to optimize current vaccine formulations and inform future vaccine efficacy and licensure studies in ruminants.