München, Univ., Diss., 2005

La capacité de Brucella suis, un microorganisme strictement aérobique, a s'adapter aux taux d'oxygène faible est un processus essentiel pour la virulence et la persistance bactérienne. Le manque d'oxygène est une condition hostile à laquelle les bactéries sont confrontées lors de la pénétration de l'hôte et pour établir leur niche replicative et la phase de persistance. Cette bactérie possède plusieurs mécanismes par laquelle elle s'adapte à cette condition. Elle peut utiliser un régulateur transcriptionel de la famille de FnrN dépendant de l'oxygène, deux cytochromes oxydases de haute affinité pour l'oxygène et une voie complète de denitrification pour résister au manque d'oxygène. Ce travail a démontré que la respiration oxydative et la denitrification peuvent être simultanément utilisés par B. suis sous microaerobiosis. RegBA, un système à deux composants chez B. suis, a été aussi identifié nécessaire dans l'adaptation bactérienne au manque d'oxygène. Ce dernier a été démontré à coordonner le contrôle des systèmes respiratoires précédemment évoqués. Un schéma de régulation global chez B. suis des voies respiratoires par le régulateur transcriptionel RegA a été suggéré : lors de la variation de l'état redox, la cytochrome bd oxidase jouerait un rôle dans la transmission d'un signal à RegB le senseur de la histidine kinase. De plus, RegA a été identifié essentiel pour la persistance de B. suis in vivo chez la souris dans les organes avec des teneurs faible en oxygène. RegA est supposé être impliqué dans l'installation de la phase de persistance bactérienne durant l'infection chronique. Cette étude a aussi identifié le rôle potentiel de RegA dans la régulation de nombreux gènes impliqués durant la phase de persistance. En utilisant une analyse transcriptomique, comparant les taux d'hybridation chez les souches sauvage et muté dans un modèle in vitro qui imite les conditions d'une infection chronique correspondant a un manque de nutriment et d'oxygène, 447 gènes avec un taux d'hybridation ≥ 2, ont été détectés réguler par RegA. Chez la souche sauvage, 45% et 55 % des gènes étaient régulés et réprimés par RegA chez la souche sauvage, respectivement. 14% des résultats du transcriptome a été choisi pour la validation génétique par RT-qPCR. RegA induit l'expression de gènes impliqués dans le métabolisme d'énergie y compris des gènes de la respiration oxidative, ce qui confirme qu'il interagit dans l'adaptation bactérienne au manque d'oxygène. RegA réprime des gènes impliqués dans la réplication d'ADN, la biogenèse de l'enveloppe et la division cellulaire, de même certains gènes dans le métabolisme d'énergie, ce qui suggère son effet sur la multiplication et l'adaptation bactérienne à l'hypoxie qui existe durant la phase de persistance. RegA a été démontré a réprimer les facteurs de virulence l'operon virB ainsi que son régulateur VjbR. De plus, cette étude a évalué le rôle de deux gènes BR1614 et BR1510 régulés par RegA et impliqués dans le métabolisme des acides gras. Dans les expériences in vivo chez la souris ont démontré que les deux gènes sont essentiels pour la survie, la multiplication et la persistance bactérienne. En conclusion, RegA régule, directement et indirectement, l'expression de gènes qui codent pour la traduction, la transcription, la production d'énergie et la conversion, la réparation d'ADN et de protéine. Ces résultats suggèrent un rôle majeur pour RegA dans la persistance bactérienne pendant la brucellose. 12% du génome de B. suis est sous le contrôle de RegA ce qui indique qu'il est un régulateur global comme son PrrA d'homologue dans Rhodobacter sphearoides. / The capacity of Brucella suis, a strictly aerobic microorganisms, to adapt to low oxygen level is of high importance as it is a required and an essential process for bacterial establishment of virulence and persistence. Oxygen deficiency is a hostile condition to which bacteria are faced when they penetrate the host and reach their replicative niche as well as the persistence phase. This bacterium possesses several mechanisms that answer remarkably to this condition. It can use an oxygen-dependent transcriptional regulator of the FnrN family, two high-oxygen-affinity terminal oxidases, and a complete denitrification pathway to resist various conditions of oxygen deficiency. This work has demonstrated that the oxidative respiration and denitrification can be simultaneously used by B. suis under microaerobiosis. RegBA, a two component systems in B. suis, was also identified to be necessary in bacterial adaptation to oxygen deficiency as it was demonstrated to coordinate the control of the respiratory systems mentioned previously. A scheme for global regulation of B. suis respiratory pathways by the transcriptional regulator RegA was suggested: under redox variation, the cytochrome bd ubiquinol oxidase would play a role in the transmission of a signal to the histidine sensor kinase RegB. RegA in addition was found to be essential for B. suis persistence in vivo in mice within low oxygenated organs. RegA is thus assumed to be involved in the establishment of bacterial persistence during chronic infections. This study also investigated the potential control of RegA in the regulation of numerous genes during the persistence phase. By using a microarray assay comparing wild-type and ∆regA mutant strains, in an in vitro model that mimic the conditions of a chronic infection corresponding to nutrient and oxygen deficiency, 447 genes with a cutoff of the level of hybridization intensities ≥2, were detected regulated by RegA. In the wild-type strain, 45% and 55 % of the genes were up-regulated and down-regulated in wildtype strain, respectively. 14% of the microarray results were selected for genetic validation by RT-qPCR. RegA induced the expression of some genes involved in energy metabolism including the oxidative respiratory genes confirming that it interacts in bacterial adaptation to oxygen deficiency. RegA down-regulated genes involved in DNA replication, cellular division cell envelope biogenesis as well as certain genes in energy metabolism suggesting its impact on bacterial multiplication and adaptation to hypoxia as it enters into the persistence phase. RegA was also found to down-regulate virulence factors such as the virB operon as well as its regulator VjbR. Moreover, this study evaluated the role of two genes BR1614 and BR1510 regulated by RegA and found implicated in fatty acid metabolism. In vivo experiments in mice demonstrated that both genes are required for bacterial survival, multiplication and persistence. In conclusion, RegA was found to regulate, directly and indirectly, the expression of genes that encode for functions in translation, general transcription, energy production and conversion, repair of DNA and protein which represent its high importance and major role in bacterial persistence during brucellosis. 12% of the genome of B. suis is under the control of RegA which makes it a global regulator such as his homologue PrrA in Rhodobacter sphearoides.

Feral hogs (Sus scrofa) are considered an exotic, free-ranging ungulate distributed within numerous countries and continents to include the United States. The reproductive efficiency, lack of predators, land use practices for domestic livestock (e.g., feeding stations, introduced water sources, intense cropping practices, etc.), and diet are leading factors in the expansion of feral hogs throughout their range. Feral hogs negatively impact floral and faunal communities, agricultural lands, and residential and recreational areas to include concerns with public safety and disease transmission. My study objectives were to (1) assess feral hog abatement strategies by (A) evaluating trap designs with the inclusion of electrical fencing, and (B) evaluating candidate baits for feral hog-specificity, and (2) assess prevalence levels for feral hog diseases. I evaluated 3 corral trap designs differing in the addition of electric fence configurations. Feral hog capture success data were collected and used to determine trap design efficacy. Treatments evaluated included (A) control corral trap with no electrical configurations, (B) corral trap with 1 electrical leg, and (C) corral trap with 2 electrical legs. ANOVA analyses suggest no differences (df = 2, P = 0.758) between trap designs; however, length of trapping effort (i.e., the number of days that trapping occurred) was a significant (df = 6, P < 0.001) factor in determining trap success. Pre-baiting was an important factor in observed trapping success. Trapping success declined after fourth day of continuous trapping. I recommend short, intensive trapping efforts (e.g., <4 days) when using corral traps in feral hog abatement programs. I also evaluated 14 candidate baits (with and without repellant) replicated 40 times to determine feral hog specificity. Three evaluated baits (i.e., PIGOUT™ strawberry, corn, and rice) were selected (df = 2, P < 0.05) more frequently by feral hogs than other combinations. Non-target species (e.g., raccoons) visited baits with repellants less (df = 2, P < 0.05) than baits without repellants. Repellant had no direct impact on feral hog visitation at bait sites. Trapping data also suggests that grains commonly farmed in local or regional areas are more likely to be consumed by feral hogs and, therefore considered in baiting options. Finally, of 412 feral hogs captured, 86 were sampled for prevalence of pseudorabies and Brucella suis. The prevalence of pseudorabies and B. suis was 20.9% and 13.9%, respectively within the study area. Based on disease study results, I recommend that natural resource managers take necessary precautions to protect themselves by wearing protective equipment and equipment and properly cooking feral hog meat. Additionally, resource managers should properly administer vaccinations to domestic and companion animals, and restricting domestic and companion animals from areas of high risk (e.g., carcasses of dead hogs and wallows).

L'accentuation des phénomènes de résistance aux antibiotiques augmente la difficulté d'enrayer les infections bactériennes. Afin de palier ce problème, la mise au point de nouvelles stratégies, telle la stratégie antivirulence, est essentielle. Ainsi, ce manuscrit propose une nouvelle approche thérapeutique contre les bactéries à développement intracellulaire. Les analyses génomiques ont permis de mettre en évidence l'histidinol déshydrogénase (HDH, EC 1.1.1.23), enzyme impliquée dans la biosynthèse de l'histidine, comme cible biologique pour la conception de nouveaux agents antibactériens. L'étude de l'inhibition de cette dernière permet une validation de l'approche sur Brucella suis, agent responsable de la brucellose, et un élargissement du spectre d'action des composés mis au point est envisagé par l'inhibition de HDH de Mycobacterium tuberculosis. Les travaux préliminaires nécessaires a cet élargissement sont présentés dans ce manuscrit. / The raise of antibiotic resistances increases the difficulty to eradicate bacterial infections. The development of new therapeutic approaches, such as the antivirulence strategy, is essential to limitate the impact of this phenomenon. This manuscript details a new therapeutic approach against intracellular pathogens. Genomic analyses allowed to discover new targets. The histidinol dehydrogenase (HDH, EC 1.1.1.23), which is an enzyme involved in histidine biosynthesis, has therefore be chosen for the conception of new antibacterial compounds. Inhibition studies of HDH of Brucella suis allows a validation of the strategy. In order to confirm the width of the therapeutic spectrum of synthesised compounds, the inhibition of HDH from Mycobacterium tuberculosis is envisaged and preliminary experiments are presented.

Livestock across the world contract several infectious diseases of both bacterial and viral origin. Swine brucellosis caused by Brucella suis and swine influenza caused by Influenza A virus affect both domestic and feral swine populations. Both the diseases have zoonotic potential to cause disease in humans with serious complications apart from inflicting huge economic losses. Infected feral swine can also act as a source of spread and outbreak where the disease is not endemic. At present, there is no vaccine available for swine brucellosis. The currently used swine influenza vaccine may not be effective against influenza strains like the recent H1N1 strain that caused a pandemic. To develop an effective bivalent vaccine for swine against these two diseases, a leucine auxotroph of the USDA approved vaccine B. abortus strain RB51 was constructed along with leuB gene complementing plasmid pNS4 to over-express antigens from Brucella and influenza. This antibiotic resistance free system over-expressed Brucella derived antigens SOD, L7/L12 and WboA in three different constructs. Against a virulent challenge of B. suis, the candidate vaccine strain over-expressing both SOD and WboA protected mice more significantly than the control group and was also found to be better protective than other candidate vaccine strains over-expressing either SOD and L7/L12 together or SOD alone. Immunoassays (ELISA) suggested that the protection afforded is Th1 type mediated immune response, as cytokine IFN-γ and IgG2a antibody sub-isotype was observed in the splenocyte culture supernatant and serum samples respectively. The strain RB51leuB platform was not expressing influenza derived antigens Hemagglutinin (HA) and Nucleoprotein (NP) when screened for expression by immunoblot. Influenza antigens, HA, NP and ectodomain of matrix protein M2e, were not found to be expressing even after optimizing their codon usage to suit Brucella tRNA preference. However, RT-PCR showed that the influenza genes mRNA were produced. In conclusion, this dissertation describes the construction of an environmentally safe antigen over-expression platform and successful employment of the system as a candidate vaccine in protecting mice against B. suis challenge. This new platform is a potential candidate for developing vaccines against other infectious diseases of livestock. This document also discusses alternate strategies for expressing influenza antigens in a Brucella platform. / Ph. D.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.