Bacterial infections are again becoming difficult to treat because the microbes are growing increasingly resistant to the antibiotics in use today. The need for novel antimicrobial compounds is urgent and to achieve this new targets are crucial. In this thesis we present a strategy for identification of such targets via a bioinformatics approach. In our first study we compared proteins with unknown and hypothetical function of the spirochete Treponema pallidum to five other pathogens also causing chronic or persistent infections in humans (Yersinia pestis, Neisseria gonorrhoeae, Helicobacter pylori, Borrelia burgdorferi and Streptococcus pneumoniae). T. pallidum was used as a starting point for the comparisons since this organism has a condensed genome (1.1 Mb). As we aimed at identifying conserved proteins important for in vivo survival or virulence of the pathogens we reasoned that T. pallidum would have deleted genes not important in the human host. This comparison yielded 17 ORFs conserved in all six pathogens, these were deleted in our model organism, Yersinia pseudotuberculosis, and the virulence of these mutant strains was evaluated in a mouse model of infection. Five genes were found to be essential for virulence and thus constitute possible antimicrobial drug targets. We have studied one of these virulence associated genes (vags), vagH, in more detail. Functional and phenotypic analysis revealed that VagH is an S-adenosyl-methionine dependent methyltransferase targeting Release factor 1 and 2 (RF1 and RF2). The analysis also showed that very few genes and proteins were differentially expressed in the vagH mutant compared to wild-type Yersinia. One major finding was that expression of the Type III secretion system effectors, the Yops, were down regulated in a vagH mutant. We dissected this phenotype further and found that the down regulation was due to lowered amounts of the positive regulator LcrF. This can be suppressed either by a deletion of yopD or by over expression of the Ribosomal Recycling Factor (RRF). These results indicate that YopD in addition to its role in translational regulation of the Yops also plays a part in the regulation of LcrF translation. We suggest also that the translation of LcrF is particularly sensitive to the amount of translation competent ribosomes and that one effect of a vagH mutation in Y. pseudotuberculosis is that the number of free ribosomes is reduced; this in turn reduces the amount of LcrF produced thereby causing a down regulation of the T3SS. This down regulation is likely the cause of the attenuated virulence of the vagH mutant. Finally, we set up a high throughput screening assay to screen a library of small molecules for compounds with inhibiting the VagH methyltransferase activity. Five such compounds were identified and two were found to inhibit VagH also in bacterial culture. Furthermore, analogues to one of the compounds showed improved inhibitory properties and inhibited the T3SS-dependent cytotoxic response induced by Y. pseudotuberculosis on HeLa cells. We have successfully identified five novel targets for antimicrobial compounds and in addition we have discovered a new class of molecules with antimicrobial properties.

Despite the large arsenal of antibiotics available on the market, treatment of bacterial infections becomes more challenging in view of the fact that microbes develop resistance against existing drugs. There is an obvious need for novel drugs acting on both old and new targets in bacteria. In this thesis we have employed a whole cell bacterial assay for screening and identification of type III secretion system (T3SS) inhibitors in Yersinia pseudotuberculosis. The T3SS is a common virulence mechanism utilized by several clinically relevant Gram-negative bacteria including Salmonella, Shigella, Pseudomonas aeruginosa, Chlamydiae and Escherichia coli. Several components in the T3SS have proved to be conserved and hence data generated with Y. pseudotuberculosis as model might also be valid for other bacterial species. We have screened a 9,400 commercial compound library for T3S inhibitors in Y. pseudotuberculosis using a yopE reporter gene assay. The initial ~ 30 hits were followed up in a growth inhibition assay resulting in 26 interesting compounds that were examined in more detail. Three of the most interesting compounds, salicylanilides, 2-hydroxybenzylidene-hydrazides and 2-arylsulfonamino-benzanilides, were selected for continued investigations. The inhibitor classes show different profiles regarding the effects on T3SS in Yersinia and their use as research tools and identification of the target proteins using a chemical biology approach will increase our understanding of bacterial virulence. The 2-hydroxybenzylidene-hydrazides have been extensively studied in vitro and show potential as selective T3S inhibitors in several Gram-negative pathogens besides Y. pseudotuberculosis. The data obtained suggest that this inhibitor class targets a conserved protein in the secretion apparatus. In cell-based ex vivo infection models T3SS was inhibited to the advantage of the infected eukaryotic cells. The salicylanilides and 2-arylsulfonamino-benzanilides have been further investigated by statistical molecular design (SMD) followed by synthesis and biological evaluation in the T3SS linked reporter gene assay. Multivariate QSAR models were established despite the challenges with data obtained from assays using viable bacteria. Our results indicate that this SMD QSAR strategy is powerful in development of virulence inhibitors targeting the T3SS.

Les bactéries du genre Shigella sont responsables de la dysenterie bacillaire, ou shigellose, chez l'être humain, causant plus de 125 millions d'épisodes et 14 000 morts par an. Cette infection est caractérisée par l'inflammation et la destruction de la muqueuse intestinale. La bactérie utilise un système de sécrétion de type III (SST3) pour manipuler la physiologie des cellules épithéliales intestinales et du système immunitaire favorisant l'invasion de la muqueuse et enrayant la mise en place d'une réponse adaptative efficace. Le SST3 peut être comparé à une seringue moléculaire traversant la paroi bactérienne sous la forme d'anneaux membranaires, contenant une tige interne (MxiI), et d’une aiguille extracellulaire (MxiH). L'assemblage de cette dernière se termine par la mise en place d'un complexe d'extrémité formé par plusieurs copies des protéines IpaD et IpaB. Le SST3 prend en charge différentes classes de substrats à sa base via un complexe protéique comprenant l'ATPase Spa47. Les translocateurs (IpaB et IpaC) sont les premiers substrats à être sécrétés. Ceux-ci sont stockés dans le cytoplasme en complexe avec leur chaperon IpgC et sont recrutés à l'extrémité de l'aiguille lors du contact avec la membrane de la cellule hôte pour y former un pore à l'aide de la protéine IpaD. Ce pore permet l'injection des autres substrats du SST3 (effecteurs) qui vont interférer avec les voies de signalisation cellulaire. Il existe deux classes d'effecteurs, les effecteurs précoces (dont OspD1) stockés au préalable dans le cytoplasme et sécrétés suite au contact cellulaire. Ce contact active l’expression des effecteurs tardifs via un couplage assuré par deux complexes, OspD1-MxiE et translocateurs-IpgC. La sécrétion d’OspD1 et des translocateurs libère leurs partenaires qui agissent comme activateurs transcriptionnels. La régulation de la sécrétion dépend de plusieurs acteurs situés dans les différentes parties du SST3. Le complexe d'extrémité et la protéine MxiC contrôlent la sécrétion aux niveaux extra- et intracellulaires alors que l'aiguille transmettrait le signal de sécrétion entre ces deux complexes. Ce paradigme reste cependant encore peu compris et le mode de fonctionnement du complexe d’extrémité et de la protéine MxiC reste à éclaircir.<p>Nos travaux menés sur la protéine IpaD nous ont permis de mettre en évidence un phénotype de sécrétion intermédiaire. Celui-ci est caractérisé par la sécrétion des translocateurs et des effecteurs précoces, sans toutefois observer de sécrétion d’OspD1 et des effecteurs tardifs, suggérant un mécanisme de discrimination entre OspD1 et les effecteurs précoces. Ce phénotype de sécrétion est similaire à celui induit par l’interaction IpaD-désoxycholate. En effet, les variants d’ipaD restant fonctionnels pour la mise en place du pore provoquent également une augmentation de l’insertion des translocateurs et du pouvoir invasif. Nous avons également identifié la région d’IpaD nécessaire au maintien d’IpaB au niveau du complexe d’extrémité ainsi qu’un rôle de son domaine central dans l’insertion du pore. Nous avons enfin étudié l’effet d’anticorps monoclonaux anti-IpaD. Ces résultats nous ont permis de proposer un modèle de fonctionnement du complexe d’extrémité lors de l’insertion du pore, d’identifier les épitopes conférant une protection in vitro et in vivo ainsi que l’existence d’un polymorphisme qui empêche la liaison de ces anticorps à IpaD provenant d’autres sérotypes.<p>Notre étude sur MxiC a mis en évidence de nouveaux partenaires d’interaction (MxiI et IpgC). Ces résultats montrent que l’interaction MxiC-MxiI est nécessaire pour la régulation de la sécrétion des effecteurs précoces par MxiC. De même, la mutation mxiIQ67A provoque un phénotype similaire à la mutation mxiHK69A, ce qui suggère que le mécanisme de régulation impliquant l’aiguille est similaire pour la tige interne. Enfin, l’interaction renforcée MxiC-Spa47, via IpgC probablement couplée à un translocateur, apporte des pistes quant au rôle de MxiC dans la sécrétion des translocateurs.<p>Les rôles identifiés pour les différents régulateurs de la sécrétion ouvrent de nouvelles pistes pour la compréhension du fonctionnement du SST3. Leurs modes de fonctionnement restent cependant encore flous et nécessitent des études complémentaires.<p><p><p>Shigella are responsible for bacillary dysentery, or shigellosis, in human beings causing over 125 million episodes and 14 000 deaths per year. This infection is characterized by inflammation and destruction of the intestinal mucosa. The bacteria use a type III secretion system (T3SS) to manipulate the physiology of intestinal epithelial cells and the immune system favoring the invasion of the mucosa and halting the development of an efficient adaptive response. The T3SS can be compared to a molecular syringe that extends from the bacterial cell wall which contains an internal rod (MxiI), and an extracellular needle (MxiH). The assembly of the latter ends with assembly of a tip complex formed by multiple copies of IpaB and IpaD proteins. The T3SS recruits different classes of substrates at its base via a complex comprising the Spa47 ATPase. The translocators (IpaB and IpaC) are the first substrates to be secreted. They are stored in the cytoplasm in complex with their chaperone (IpgC) and are recruited at the needle tip upon contact with host cell membrane to form a pore via IpaD. This pore allows the injection of other substrates of the T3SS (effectors), which will interfere with the cellular signaling pathways. There are two classes of effectors, early effector (including OspD1) stored in the cytoplasm and secreted upon cell contact. This contact activates the expression of late effectors genes through a complex formed by MxiE (blocked by OspD1) and IpgC. Both proteins are released through OspD1 and translocators secretion. Secretion regulation depends on several actors located at different parts of the T3SS. The tip complex and the gatekeeper MxiC regulate secretion at the T3SS tip and base, the needle subunits transmitting a secretion signal between these two complexes. This paradigm, however, is still poorly understood and the operating mode of the tip complex and MxiC remains unclear.<p><p>Our work on IpaD protein allowed us to identify an intermediate secretion phenotype which is characterized by the secretion of translocators and early effector, but no secretion of OspD1 and late effectors, suggesting a discriminating mechanism between early effectors and OspD1. This secretion phenotype is similar to that induced by deoxycholate-IpaD interaction. Indeed, IpaD point mutants responsible for this phenotype cause an increase in the pore insertion and cell invasion. We also identified the region of IpaD necessary to maintain IpaB at the needle tip as well as a role of IpaD central domain in the pore insertion. We finally studied the effect of anti-IpaD monoclonal antibodies. These results allowed us to propose a working model of the tip complex end upon pore insertion, identify epitopes conferring protection in vitro and in vivo as well as the existence of a polymorphism that prevents the binding of these antibodies to IpaD from other serotypes.<p><p>Our MxiC study showed new interaction partners (MxiI and IpgC). These results showed that the MxiC-MxiI interaction is necessary for the regulation of early effectors secretion of by MxiC. Moreover, a mxiIQ67A mutation causes a phenotype similar to the mutation mxiHK69A, suggesting that the regulatory mechanism involving the needle is shared by the inner rod. Finally, the enhanced interaction MxiC-Spa47 through IpgC, probably in complex with a translocator, provides clues for the role of MxiC in translocators secretion.<p><p>The roles identified for the various regulators of secretion open up new avenues for understanding how the T3SS functions. Their ways of working are however still unclear and require further study.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

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Burkholderia cenocepcia is one of the serious infectious agents of respiratory tract among cystic fibrosis patients. There are problems mainly with strains which are capable of epidemic spread. The known epidemic in the Czech Republic was caused by ST32 strain in the past. In this work, there was completed whole genome sequence of referential isolate 1232 of B. cenocepacia ST32 in cooperation with bioinformatics by new generation sequencing techniques and by determining the problematic areas by a combination of Sanger sequencing bioinformatics approaches and manual assembling of sequence reads localized in these areas. The final version of the genome sequence was annotated by PGAAP and at the present time it is finalized. Second part of this work is dedicated to looking for a prognostic marker of infection caused by ST32 strain in patients with cystic fibrosis. We analysed the results of ST32 trancriptomic experiment and chose genes possibly connected with the cepacia syndrome - serious, mostly fatal state of infection. By quantitative PCR we compared their expression in isolates from 4 patients from time of cepacia syndrome and month before that. We checked the possibility of direct detection of the expression of these genes in clinical material. We identified genes for type III secretion system as...

Pseudomonas aeruginosa est un bacille gram négatif, ubiquiste de l'environnement. Ce pathogène opportuniste humain provoque sous sa forme planctonique des infections aiguës dont le facteur de virulence clé est le SST3. P. aeruginosa peut aussi se développer sous forme de biofilm où elle exprime entre autre le SST6-1 et induire des infections chroniques. L'expression ciblée des différents facteurs de virulence est liée à l'intégration de nombreux stimuli environnementaux transduits au moyen de systèmes à deux composants ou encore de messagers secondaires, comme le di-GMPc et l'AMPc, et conduisant à une régulation très fine, conférant une grande capacité d'adaptation à la bactérie. La nature, de même que les mécanismes impliqués dans la transduction du signal, n'ont pas encore été tous identifiés à ce jour. Le but de cette thèse était de décrypter ces mécanismes moléculaires de détection et de transduction du signal gouvernant la réponse adaptative de ce pathogène à son environnement au moyen de différentes approches : chémogénomique, mutagénèse aléatoire et d'étude d'isolat clinique. Lors de cette thèse, nous avons criblé deux chimiothèques commerciales à la recherche de molécules activatrices du SST3 et du SST6-1 et nous avons pu établir un test de criblage à haut débit robuste pour les criblages de plus larges banques de composés. En utilisant une souche avec deux rapporteurs, nous avons réalisé une banque de mutants par transposons et nous avons trouvé des mutants affectés dans leur expression du SST3, candidats intéressants pour identifier de nouveaux régulateurs du système. Enfin, grâce à l'analyse de l'isolat clinique CHA (issu d'un patient atteint de mucoviscidose), nous avons découvert qu'une délétion dans le gène codant pour le régulateur majeur GacS définissait le phénotype agressif de cette souche. / Pseudomonas aeruginosa is a gram negative bacillum present in several places. This opportunistic pathogen has the capacity to infect a wide range of hosts: plants, animals, humans. This bacterium, that shows an impressive adaptability relying on a multifactorial virulence, possesses two lifestyles. These lifestyles are associated with specific virulence patterns of expression. Under its planktonic form, P. aeruginosa can provoke acute infections thanks to the activation of T2 and T3SS or induces chronic infections in cystic fibrosis patients' lungs where it establishes a biofilm (communautary life). The expression of virulence factors is linked to the integration of several environmental cues that are transduced through two-component systems and secondary messengers like c-di-GMP and that lead to a fine tuned regulation. The nature and the mechanisms involved in this signal transduction remain largely unknown. The goal of this thesis was to decipher molecular mechanisms of signal detection and transduction that govern the adaptive pathogen response to host environment using the combination of a chemogenomics, random mutagenesis and study of clinical isolate. During this work, we screened two commercial libraries and set up a robust high throughput screening test to analyse huge molecules libraries. By setting up a double reporter-gene strain, we realized a transposon mutagenesis bank and identified interesting candidates with a down-regulated T3SS. Finally, the study of the particular clinical isolate CHA (from a cystic fibrosis patient), leads to the discovery that a deletion in the gene encoding for the important regulator GacS shapes the aggressive phenotype of this strain.

Bordetella pertussis is a Gram-negative strictly human pathogen and the major causative agent of whooping cough or pertussis. The incidence of this highly contagious respiratory disease in developed countries has increased in the last decades. One of the less characterized virulence factors of B. pertussis is the type three secretion system (TTSS) which is responsible for the secretion of the effector proteins into host eukaryotic cells. This diploma thesis sheds light onto factors influencing TTSS in vitro activity. Although TTSS of laboratory strain Tohama I was induced by biologically active compounds present in blood (e. g. complement proteins), TTSS of recent clinical isolate B1917 seems to be induced permanently. Furthermore, BB0302 encoding a GntR family transcription regulator in B. bronchiseptica RB50 (homologous to BP0209 of Tohama I) was studied, however, the deletion of this gene did not affect the TTSS functionality. Serum resistance is a factor that plays a key role in the pathogenesis of B pertussis. We show that Czech recent isolates (2008-2015) are significantly more resistant to serum killing in vitro than the original vaccine strains (1954-1965). This phenomenon seems to result from the adaptation of global B. pertussis population to its human host. In addition, this diploma...

<p>Bacteria employ virulence mechanisms to promote fitness that are generally detrimental to a host organism. The Gram-negative pathogen <em>Salmonella enterica </em>utilizes type three secretion systems (T3SS) to inject proteins termed effectors into the host cell cytoplasm where normal cellular function is modified. The coordinated T3SS assembly, and delivery of effectors to the cytoplasmic face of the T3SS is aided by virulence chaperones. The interaction of effector-chaperone complex with the T3SS occurs via an ATPase protein, where the complex is dissociated and the effector is unfolded, presumably for passage through the T3SS. The virulence chaperone network associated with the <em>Salmonella </em>pathogenicity island two (SPI-2) encoded T3SS has not been fully characterized. Additionally, the T3SS ATPase protein encoded within SPI-2, SsaN, has yet to be examined for functional motifs or a precise role in effector secretion. The contents of this thesis describe the characterization of two novel virulence chaperones, SrcA and SscA, and the T3SS ATPase SsaN. SrcA is a virulence chaperone for the effector substrates SseL and PipB2, and adopts the characteristic horseshoe-like structure common amongst effector chaperones. SscA is a chaperone for the translocon component SseC of the T3SS structure, and both proteins impact the regulation of SPI-2 promoters. The structure of SsaN resembles other T3SS ATPases, although different conformations exist between the structures, potentially highlighting regions with T3SS function. Additionally, an N-terminal domain was found to be dispensable for membrane localization, and residues within the predicted hexamer model impact effector secretion. These results identify novel virulence chaperones essential for T3SS function, and characterize the T3SS ATPase protein encoded within SPI-2. These findings greatly expand our knowledge of the virulence mechanisms utilized by <em>S. enterica</em>.</p> / Doctor of Philosophy (PhD)

La bactérie phytopathogène E. amylovora, est l'agent responsable du Feu bactérien des Spiraeoideae (pommier, poirier, pyracantha), une maladie caractérisée par l'apparition de symptômes nécrotiques des tissus infectés. Le pouvoir pathogène d’E. amylovora repose entre autre sur un système de sécrétion de type III (SSTT) qui permet la sécrétion et l'injection d'effecteurs dans la cellule hôte végétale. Parmi les protéines injectées par le T3SS d'E. amylovora, DspA est essentielle au pouvoir pathogène de la bactérie puisqu’un mutant dspA est non pathogène sur plante (Gaudriault et al., 1997). Le rôle de DspA est dual, d’une part, l’expression de dspA est suffisante pour provoquer des symptômes nécrotiques sur plante et une toxicité chez la levure, d’autre part, DspA est impliquée dans la suppression des réactions de défense telles que la déposition de callose (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA appartient à la famille des effecteurs AvrE qui sont répandus chez les bactéries phytopathogènes et semblent posséder une fonction similaire. Cependant, peu de connaissance existe sur la structure ainsi que la fonction de DspA. L'objectif de ce travail de thèse était de déterminer les domaines ou motifs importants pour la fonction de DspA. Pour cela nous avons choisi d'effectuer une analyse in silico et fonctionnelle de la protéine DspA. L'analyse in silico révèle la présence d'un domaine bêta-propeller au sein de la protéine DspA ainsi que de tous les homologues analysés. De plus, l'analyse fonctionnelle indique que ce domaine est important pour la structure et la fonction de DspA. Dans un second temps, j'ai étudié le mécanisme d'action de DspA dans la levure Saccharomyces cerevisiae. J'ai pu mettre en évidence que l'expression de dspA chez la levure induit un arrêt de croissance et une forte altération du trafic cellulaire. L'étude de mutants de levure suppresseurs de la toxicité de DspA, effectuée avant mon arrivée au laboratoire, montre que les suppresseurs les plus forts sont affectés dans la voie de biosynthèse des sphingolipides, je me suis donc plus particulièrement intéressée au rôle des sphingolipides dans la toxicité générée par DspA. Nos résultats montrent que DspA inhibe la biosynthèse des sphingolipides indirectement via les régulateurs négatifs de la voie, les protéines Orms. / Erwinia amylovora is the causative agent of fire blight of Spiraeoideae (apple, pear, pyracantha), a disease characterized by the apparition of necrotic symptoms on infected tissues. The pathogenicity of E. amylovora relies on a functional type III secretion system (T3SS) that allows secretion and injection of effector proteins into the host plant cell. Among these effector proteins injected by the T3SS of E. amylovora, DspA is essential to the bacteria disease process since a dspA mutant is nonpathogenic on plants (Gaudriault et al., 1997). DspA has a dual role; on the one hand dspA expression is sufficient to induce cell death on plants and toxicity on yeast, on the other hand, DspA is involved on suppression of defense reactions like callose deposition (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA belongs to the AvrE familly of type III effectors which are widespread on phytopathogenic bacteria and likely possess a similar function. However, the structure and function of DspA remain unknown. In the first part of my thesis, I attempted to characterize domains or motifs important for the function of DspA. We performed an in silico and a functional analysis of the DspA protein. In silico analysis predicted a bêta-propeller domain in DspA and all the analysed effectors. In the second part of my thesis, I analysed the mechanism of function of DspA in the yeast Saccharomyces cerevisiae. Results showed that expression of dspA in yeast inhibits cell growth and alters the actin cytoskeleton and endocytosis. Screening of the Euroscarf library for mutants resistant to DspA induced toxicity revealed that mutants impaired in the sphingolipid biosynthetic pathway are the best suppressors. Based on this results, I attempted to determine the role of sphingolipids in the toxicity induced by DspA. Results showed that DspA inhibits indirectly the sphingolipid biosynthetic pathway via the negative regulators, Orm proteins.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012