Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Tuberculosis is a re-emerging global health problem. Bacille Calmette-Guerin (BCG), the available vaccine against the disease, is only effective short term and is associated with adverse reactions clinically. The development of new effective vaccines will require an understanding of virulence, immunogenic factors and the beneficial immune responses induced in the human host. My thesis investigates phoP and whiB3, two genes associated with virulence and immunogenicity in Mycobacterium tuberculosis. Study of PhoP in a natural phoP mutant, BCG-Prague, and in the clinically safe BCG-Japan, shows that over-expression of PhoP increases the immunogenicity of these vaccine strains. In addition, I found that WhiB3 impacts carbon metabolism in BCG-Birkhaug and BCG-Sweden, although the effect of this on virulence in vivo is still unclear. The characterization of genes involved in virulence and immunogenicity allows us to develop novel approaches for improving the efficacy of BCG, which has important implications for future TB vaccine development.

Les phytases hydrolysent les phytates représentant la forme majeure de stockage du P dans les céréales. Ces phytates sont aussi des facteurs anti-nutritionnels qui chélatent les cations réduisant leur absorption. Dans le premier volet de cette thèse, une nouvelle souche bactérienne produisant une phytase extracellulaire a été isolée et identifiée comme Bacillus amyloliquefaciens US573. L’enzyme «PHY US573» a été purifiée et caractérisée en comparaison avec deux phytases commerciales Ronozyme PL et Natuphos. PHY US573 se distingue par sa forte thermostabilité en présence de calcium. En outre, PHY US573 se caractérise aussi par une tolérance remarquable aux sels comme le NaCl et LiCl. L’ensemble de ces propriétés montre que PHY US573 pourrait être une candidate intéressante pour des applications en alimentation animale ou en agriculture pour améliorer la biodisponibilité du P-phytique pour les plantes. Dans le deuxième volet, la souche Streptomyces sp. US42 produisant une activité phytase extracellulaire a été sélectionnée. L’enzyme «PHY US42» a été purifiée et caractérisée. PHY US42 est calcium dépendante également une grande stabilité en présence de sels biliaires et des protéases digestives. La modélisation moléculaire de PHY US42 indique qu'elle appartient au groupe des β-propeller phytases qui sont généralement calcium-dépendantes. Vu ses propriétés biochimiques intéressantes, PHY US42 constitue une bonne candidate comme additif dans les aliments pour animaux monogastriques en combinaison avec une histidine acide phytase. Enfin dans un troisième volet, nous nous sommes intéressés à l’étude de la régulation de l'expression du gène phytase de S. coelicolor M145 (sco7697) chez S. coelicolor M145, S. lividans TK24 ainsi que chez ses deux mutants ppk et phoP. Ainsi, en plus des boites pho localisées en amont de la région promotrice -35 siège de la régulation positive PhoP-dépendante, nous avons révélé pour la première fois que la RD localisée en aval de la région promotrice -10 est le siège d’une forte régulation négative par un répresseur inconnu. Ce dernier empêcherait l’activation PhoP-dépendante de l’expression du gène phytase. / Phytases hydrolyse phytate representing the major storage form of P in cereal. phytates are also anti-nutritional factors that chelate cations such as Ca²⁺, Mg²⁺, Fe²⁺, Z²⁺ reducing their absorption. The low bioavailability of phytic phosphorus in monogastric animals require their food supplementation with Pi to meet the needs of the animal in P. This creates an extra cost and increases the environmental pollution by the manure excretion highly charged phosphate. In the first part of this thesis, from soil samples taken near hot hydrothermal waters of the region Elhamma in southern Tunisian, a new bacterial strain producing extracellular phytase was isolated and identified as Bacillus amyloliquefaciens US573. The enzyme referred "PHY US573" was purified and characterized in comparison with two commercial acid histidine phytases Ronozyme PL and Natuphos. PHY US573 is calcium dependent and has an optimum activity at pH 7.5 (5 for Ronozyme and 5.5 for Natuphos) and 70°C (55°C for Ronozyme and Natuphos). PHY US573 is distinguished by its high thermostability, in fact, it keeps 93% of its activity after incubation for 10 min at 75°C in the presence of calcium while Ronozyme and Natuphos keep only 45% and 53% of their activity, respectively. This enzyme is specific for phytic acid and also has a very good stability at pH 3 to 9 and a perfect stability in presence of bile salts. In addition, PHY US573 is also characterized by a remarkable salt tolerance because it retains 80 to 95% of its activity in the presence of 20 g/l of NaCl and LiCl, respectively. All these properties shows that PHY US573 could be an interesting candidate for applications in feed industry alone or in combination with an histidine acid phytase. In a second part of this thesis, from the Streptomyces collection of LMB-CBS, a strain producing extracellular phytase activity was selected and identified as Streptomyces sp. US42. The enzyme "PHY US42" was purified and characterized. PHY US42 has a calcium-dependent activity (such as Bacillus phytases), optimally active at pH 7 and 65°C. PHY US42 is perfectly stable at pHs ranging from 5 to 10 and its thermal stability is greatly increased in the presence of calcium. Indeed, PHY US42 maintains 80% of its activity after 10 min of incubation at 75 °C in the presence of calcium. PHY US42 has also a high stability in the presence of bile salts and digestive proteases. Molecular modeling of PHY US42 indicates that it belongs to the β-propeller phytase group which are usually calcium-dependent. Given its interesting biochemical properties, PHY US42 which would operate mainly in the intestine, is a good candidate for use as an additive in agastriques fish food or in combination with an histidine acid phytase in feed industry. Finally in a third part, we are interested in studying the regulation of the expression of the phytase gene of S. coelicolor M145 (sco7697) in S.coelicolor M145, S.lividans TK24 and among its two mutants ppk and phoP. To do this, we merged the wild promoter regions (phyWT) or mutated (phym1, phym2, phym1+2) of sco7697 gene with the GUS reporter gene encoding ß-glucuronidase activity. Thus as expected, we demonstrated that the deletion of the PHO box located upstream of the -35 reduces the level of induction of sco7697 in conditions of Pi limitation. Moreover, we have revealed for the first time that the alteration of RD located downstream of -10 correlates with a dramatic increase of GUS expression when PhoP is present. Our results demonstrate that this RD is the seat of a strong negative regulation by an unknown repressor. This would prevent the PhoP-dependent activation of expression of the phytase gene.

<p>  </p> <p>PhoQP is a two-component system that regulates the transcription of ~5% of the genes of <em>Salmonella enterica</em>. The membrane-bound PhoQ protein is phosphorylated in response to low extracellular Mg²⁺ concentration, acid pH, and a number of antimicrobial peptides. The inorganic phosphate bound to PhoQ is transferred to PhoP, which according to the classical model, acts as a typical transcriptional activator of its target genes. However, Will et al. (doi.org/10.1038/ncomms6270) proposed an alternate “counter-silencing” model, according to which genes in the PhoP regulon that were acquired by <em>Salmonella</em> via horizontal transfer are repressed by the generalized DNA-binding protein H-NS at high [Mg²⁺] and are induced at low [Mg²⁺] because the phosphorylated PhoP displaces the H-NS from the promoters and lifts repression. We evaluated this model by examining the transcriptional regulation of the <em>mgtCBRU-cigR </em>operon, which encodes the virulence protein MgtC and the Mg²⁺ transport protein MgtB and is in the SPI-3 pathogenesis island that has been acquired by <em>Salmonella</em> via horizontal transfer. Our main finding was that in the non-pathogenic strain of <em>S</em>. Typhimurium (LT2), induction of the <em>mgtCBRU-cigR</em> operon by Mg²⁺ limitation requires a functional PhoP protein, regardless of the presence or absence of H-NS. Interestingly, the pathogenic strain of <em>S</em>. Typhimurium (ATCC 14028s) revealed PhoP-independent transcription in the absence of H-NS, but only under inducing conditions. Thus, our results do not support the counter-silencing model and are consistent with the canonical view that PhoP is needed as a transcriptional activator of genes in the PhoP regulon.</p>

Salmonella spp. sind fakultative intrazelluläre Pathogene, die gastrointestinale und systemische Erkrankungen in einem umfassenden Wirtsbereich, einschließlich Tier und Mensch, hervorrufen. Salmonella benötigt verschiedene Virulenzgene für die Infektion welche auf sogenannten Salmonella Pathogenitäts-Inseln (SPI) kodiert sind. Hinzu kommt, dass auch zahlreiche im Salmonella Genom verstreuten Gene an verschiedenen Aspekten von Virulenz und Pathogenese beteiligt sind. In der vorliegenden Studie wurde die Funktion eines zuvor nicht beschriebenen putativen transkriptionellen Regulators (STM0029) charakterisiert und definiert. Dieser scheint für die Abwehr von zellulären bakterizid wirkenden Verbindungen und das Überleben des Bakteriums innerhalb einer intrazellulären Nische von entscheidender Bedeutung zu sein. Die STM0029-deletierte Mutante wies eine gesteigerte Sensitivität gegenüber antimikrobiellen Peptiden und bakteriziden Verbindungen auf. Dazu zählten α-Defensin-1, β- Defensin-1, β-Defensin-2, LL-37 und Polymyxin B sowie Komponenten des Komplementsystems. Unerwartet war die Beobachtung, dass die Expression von STM0029 durch das PmrA/B Zwei Komponenten System reprimiert vorlag, während das PhoP/Q Zwei Komponenten System keinen Einfluss auf die Expression von STM0029 zu scheinen hat. Beide Komponent Systeme spielen bekanntlich eine entscheidende Rolle bei der Expressionsregulation von Genen die für das intrazelluläre Überleben von Salmonella wichtig sind. Bemerkenswert ist, dass ein Set von Genen welche an der Biosynthese und/oder der Modifikation für das LPS O-Antigen sowie des Peptidoglykans in der bakteriellen Zellwand beteiligt ist, im STM0029 Deletionshintergrund herab reguliert vorlag. Dieses Ergebnis deutet darauf hin, dass das STM0029 Genprodukt die Persistenz des Pathogen in Wirtszellen beeinflusst. Möglicherweise geschieht dies durch das Umgehen von wirtseigenen Abwehrmechanismen. / Salmonella spp. are facultative intracellular pathogens, which cause gastrointestinal and systemic diseases in a broad range of hosts including animals and humans. In addition to virulence genes clustered within pathogenicity islands, numerous additional genes scattered throughout the genome are also involved in various aspects of Salmoenlla virulence and pathogenesis. In this study, I identified a Salmonella putative transcriptional regulator encoded by a previously uncharacterized open reading frame designated STM0029. Deletion of STM0029 altered the expression of genes involved in both the resistance to host bactericidal challenges, and bacterial cell wall biosynthesis in S. Tyhpimurium. The ΔSTM0029 strain showed a defect in the resistance to host antimicrobial peptides, including α-defensin-1, β-defensin-1, β-defensin-2, LL-37, and polymyxin B as well as serum challenges compared to the wildtype. Unexpectedly, expression of STM0029 was found to be repressed by the PmrA/B two component system, but appeared to be independent of the PhoP/Q two component system, both of which are well-known regulatory systems involved in the regulation of expression of genes involved in Salmonella intracellular survival. Notably, the expression of a set of genes involved in bacterial LPS O-antigen and peptidoglycan biosyntheses and modifications showed decreases in the absence of STM0029. These experimental results indicate that the STM0029 gene product in S. Typhimurium contributes to resistance against host cell defense mechanisms, likely through regulation of genes involved in LPS O-antigen and peptidoglycan biosynthesis and modifications.

Viele Bacillus Stämme sekretieren Phytasen, diese katalysieren die Dephosphorylierung von myo-Inositolhexakisphosphat (phytate). Das monocystronische phyC Gen aus dem im Boden lebenden Bacillus amyloliquefaciens FZB45 konnte als Teil des, durch Phosphatmangel induzierbaren, PhoPR-Regulons identifiziert werden. Der Transkriptionsstart des SigmaA-abhängigen Promotors wurde 27 bp upstream von Translationsstart bestimmt. Der Promotor weist jedoch eine ungewöhnliche Struktur auf, da die –35 und die –10 Region durch ein 21 bp Fenster voneinander getrennt sind. Die in vitro Transkriptionsanalyse zeigte, dass PhoP-P für die Initiation der phyC-Transkription notwendig ist. Die PhoP-Bindungsstellen der meisten B. subtilis Promotoren, die durch PhoP aktiviert werden, besitzen mindestens vier TTAACA-ähnliche Sequenzmotive, welche durch 5-6 bp- Intervalle getrennt sind. Die upstream Region von phyC aus B. amyloliquefaciens FZB45 weicht von dieser Struktur ab. Hier konnten nur zwei solcher Motive, die für die Bindung eines PhoP-Dimeres zuständig sind und die Positionen -47 und -35 überlagern, identifiziert werden. Ein weiteres Motiv befindet sich zwischen -13 und –8 und überlappt um eine Base mit der –10 Region. Die Funktionen der drei Bindungsstellen konnten durch DNaseI-Footprinting ermittelt werden. Ein PhoP-Dimer besetzt die –35 Region und dirigiert dabei die RNA-Polymerase wahrscheinlich in Richtung –10 Region. Durch die Bindung von PhoP an die PhoP-Erkennungsstelle bei –10 wird die Transkription gehemmt. Diese PhoP-vermittelte duale Kontrolle des phyC-Promotors scheint bis lang einzigartig für Pho-Regulongene zu sein. Der globale Regulator AbrB konnte als weiterer Repressor des phyC identifiziert werden. Die Bindungsstelle befinden sich upstream von –147 und downsteam von +29, müssen aber in weiteren Experimenten genauer spezifiziert werden. Diese Arbeit stellt als erste die Modelle der phyC-Regulation durch PhoP und AbrB vor. / Several Bacillus strains secrete phytase, an enzyme catalysing dephosphorylation of myo-inositol hexakisphosphate (phytate). The monocistronic phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 was identified as a member of the phosphate-starvation inducible PhoPR regulon. The transcriptional start was determined downstream of a sigmaA-like promoter region located 27 bp upstream of the translation start codon. Inspection of the phyC promoter sequence revealed an unusual structure, since the -35 and -10 region are separated by a window of 21 bp. In vitro transcription analysis established that PhoP-P is necessary to initiate the transcription from phyC promoter. PhoP binding boxes occurring in most B. subtilis promoters activated by PhoP consist of at least four TTAACA-like sequences repeated at intervals of 5-6 bps. The upstream region of the B. amyloliquefaciens FZB45 phyC gene deviates from this general architecture in that there is only one appropriate binding site for the dimeric PhoP protein, which consists of two motives centered at -47 and -35 and separated by five base pairs. A single PhoP binding site is located at -13 to -8, nearly matching the -10 consensus. Functionality of the three PhoP binding boxes was demonstrated by DNaseI footprinting suggesting that a pair of dimeric PhoP molecules cover the -35 region directing the RNA-polymerase to the –10 region. Binding of PhoP at a single PhoP binding site covering the -10 consensus repress the transcription. It seems to be a unique feature of the phyC promoter structure and has not been reported for any other member of the PhoPR regulon, previously. Furthermore an inhibitory effect via AbrB, a global regulator, could be verified. The binding regions could be determine upstream of –147 and downstream of +29, but have to be specify in further experiments. This work presents for the first time the models for the regulation of phyC in Bacillus via PhoP and AbrB.

The global increasing problem of antibiotic resistance necessarily drives the pursuit and discovery of new antimicrobial agents. Antimicrobial peptides (AMPs) initially seemed like promising new drug candidates. Already members of the innate immune system, it was assumed that they would be bioactive and non-toxic. Their common trait for fundamental, non-specific mode of action also seemed likely to reduce resistance development. In this thesis, we demonstrate the ease with which two species of pathogenic bacteria, the gram-negative Salmonella typhimurium (S. typhimurium), and the gram-positive Staphylococcus aureus (S. aureus), can gain increased tolerance and stable resistance to various AMPs. By serially passaging each bacterial species separately under increasing AMP selection pressure we observed increasing AMP tolerance. Resulting in independent bacterial lineages exposed to four different AMPs (including a two-AMP combination) that exhibited 2 to 16-fold increases in MIC. Substantial cross-resistance between the AMPs was observed. Additionally, the S. aureus mutants were found to be cross-resistant to human beta-defensins 1, 2, 3, and 4. The LPS molecule, with mutations in the waaY, pmrB and phoP genes, was the principal target for S. typhimurium resistance development. The main target for S. aureus remained elusive. Reduced membrane potential was a common change for two of the mutants, but not for the others. All sequenced mutants had one or more mutations in various stress response pathways. Fitness of the resistant mutants was assayed by growth rate analysis and in vitro virulence factor testing (e.g. survival response to bile, superoxide, acidic pH). Furthermore an in vivo survival/virulence test involving a mouse competition experiment (S. typhimurium) and sepsis model (S. aureus) was performed. In the absence of AMPs there was often little or no fitness reduction in the mutants. Our results suggest that AMP resistance mechanisms do not irrevocably weaken either species with regard to virulence characteristics or survival within the host. In light of these findings, we suggest that the progression of therapeutic use of AMPs should proceed with great caution since otherwise we might select for AMP resistant mutants that are more resistant to our innate host defenses and thereby potentially more virulent.

No description available.

Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.