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Zatakia, Hardik M. "Characterization of symbiotically important processes in Sinorhizobium meliloti." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/56652.
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Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis.
Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed.
Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis.
Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low
abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria.
These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers.<br>Ph. D.
2
Berry, Jamie. "Structural characterization of type IV pilus biogenesis proteins." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/structural-characterization-of-type-iv-pilus-biogenesis-proteins(1e0d7119-58d5-4e5d-839d-daef8deb76ab).html.
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Type IV pili, or fimbriae, are long, thin proteinaceous appendages found on the surface of many well-known pathogens. They mediate a variety of important virulence functions for the organism, such as twitching motility, biofilm formation, uptake of genetic material and host cell recognition and adhesion. Pili are formed by the rapid polymerization and de-polymerization of the pilin subunit, and this is orchestrated by a complex macromolecular machine which spans the bacterial cell envelope, requiring a variety of gene products. The type IV pilus biogenesis system is closely related to the bacterial type II secretion system, one of six designated multi-protein cell envelope complexes which are dedicated to the specific secretion of exotoxins and virulence factors. Many of these secretion systems also produce fimbrial structures to facilitate the extrusion of their substrates or to communicate with the host. As they form crucial virulence factors, the secretion systems and the type IV pilus biogenesis system have become attractive potential antimicrobial targets and obtaining structural and functional information for the components of these systems is an important first step towards achieving this.Type IV pili appear on the surface of bacteria through an outer membrane pore, PilQ, which is a member of the secretin family. Secretins are also found in the type II and III secretion systems, but the way in which they are regulated remains unclear. PilQ forms a dodecameric chamber in the outer membrane with a large vestibule which reaches into the periplasm, composed of its N-terminal domains. In this project, N-terminal domains from PilQ were produced in recombinant form and their structures determined by NMR. One of these domains revealed an eight-stranded beta-sandwich structure which appears to be unique to type IV pilus secretins and has not been structurally characterized before. Another revealed an alpha/beta type fold which is common to secretins of other systems. In the second part of this project, the interaction formed between the N-terminal alpha/beta domains of PilQ and an essential inner membrane-anchored lipoprotein, PilP, was probed by NMR chemical shift perturbation. Based on changes to the 15N-HSQC spectra the binding site was mapped onto each protein to produce a computational model for the complex formed between the two. Using a recent cryo-EM structure for the Neisseria PilQ dodecamer determined by colleagues, it was possible to model the PilQ N-terminal domains in complex with PilP into the electron density map. This produced a model for the trans-periplasmic assembly formed by PilQ and PilP in the type IV pilus biogenesis system, and led to the conclusion that the PilQ dodecamer needs to disassemble considerably at the base to accommodate a pilus fibre. The novel beta-domains might therefore function to gate or open the secretin, and PilP may play a role in stabilizing the secretin during this and serve to connect the outer and inner membrane system components.
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Jacobsen, Theis. "Structure and assembly of bacterial type IV filaments unravelled by an integrative approach." Electronic Thesis or Diss., Sorbonne université, 2022. http://www.theses.fr/2022SORUS146.
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La superfamille des filaments de type IV (TFF) est un groupe de machines moléculaires localisées dans la membrane des bactéries et des archées. Ces machines associent des polymères protéiques de manière non-covalente appelés des pili, qui s’étendent depuis la cellule pour réaliser plusieurs fonctions qui ont évolué spécifiquement pour s’adapter à des organismes hôtes différents. La superfamille TFF comprend le système de sécrétion de type II (T2SS) et les pili de type IVa (T4aP). Le T2SS induit la sécrétion de substrats chez les bactéries Gram-négatives. Ces substrats sont en général des enzymes qui dégradent les complexes carbohydrates, le peptidoglycane, les lipides, ce qui à terme entraîne la libération de nutriments. Les T4aP sont de longues fibres flexibles qui sont ancrées dans la membrane et permettent de nombreuses fonctions. Le mécanisme par lequel le T2SS et les T4aP remplissent ces différentes fonctions n’est toujours pas entièrement compris. Pour comprendre le mécanisme de sécrétion du T2SS, nous avons étudié par RMN la structure de la pseudopiline OutG, le composant majeur du pseudopilus chez Dickeya dadantii. Dans une seconde partie, nous avions pour objectif d’aborder la structure et l’assemblage des pilines mineures, des protéines qui composent le T4P d’Escherichia coli entérohémorragique. Nous avons optimisé la surexpression, la purification et le marquage de les pilines mineures pour leur étude par RMN. De plus, la modélisation des pilines et le cross-linking ont été réalisés sur les échantillons de pseudopili T4P et T2SS purifiés en tant que méthodologie pour déterminer la structure et les interactions des pilines et pseudopilines au sein du pilus natif<br>The type IV filament (TFF) superfamily is a group of molecular machineries located in the membrane of bacteria and archaea. These machineries assemble non-covalent protein polymers called pili extending away from the cell to perform multiple functions which have evolved specifically to adapt to different host organisms. The TFF superfamily includes the type II secretion system (T2SS) and the type IVa pili (T4aP). The T2SS promotes the secretion of substrates in Gram-negative bacteria. These substrates are in general enzymes degrading complex carbohydrates, peptidoglycan, and lipids, resulting in the release of nutrients. The T4aP are long flexible fibres anchored in the membrane and enable various functions such as twitching motility, DNA uptake and biofilm formation. The mechanism by which the T2SS and T4aP pilus fulfil their different functions is still not completely understood. To understand the mechanism of secretion by T2SS, we studied the structure of the pseudopilin OutG, the major component of the pseudopilus in Dickeya dadantii by Nuclear Magnetic Resonance (NMR). In a second part, we aimed to address the structure and the assembly of minor pilins, protein components of Enterohemorrhagic Escherichia coli T4aP. We optimised the overexpression, purification and labelling of the minor pilins for their structural study by NMR. Furthermore, molecular modelling of the minor pilins and crosslinking mass spectrometry were performed on whole T4aP and T2SS pseudopili purified samples as a methodology to determine the structure and the interactions of pilins and pseudopilins within the native pilus
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Luna-Rico, Areli-Noemi. "Enterobacterial type IV pili : structure, assembly and molecular function." Thesis, Sorbonne Paris Cité, 2018. https://theses.md.univ-paris-diderot.fr/LUNARICO_Areli_4_va_20180629.pdf.
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Nombreuses espèces bactériennes présentent des fibres à leur surface qui leur permettent d’interagir avec leur environnement. Les pili de type 4 (PT4) sont des fibres longues, fines et flexibles, impliquées dans des fonctions multiples telles que l'adhérence, la motilité, la sécrétion, l'import d'ADN et la formation des biofilms. Ils sont composés de milliers de copies de sous-unité majeure de piline et sont assemblés par un complexe protéique localisé dans l'enveloppe bactérienne. Dans cette étude, nous nous sommes intéressés aux PT4 chez Escherichia coli entérohémorragique (EHEC) de sérotype O157: H7. EHEC est un agent pathogène humain d'origine alimentaire qui provoque des épidémies de diarrhées hémorragiques et/ou des atteintes rénales sévères appelées syndrome hémolytique et urémique (SHU), qui peuvent être mortelles. Les PT4 chez EHEC sont composés de la sous-unité majeure PpdD et probablement des pilines mineures PpdA, PpdB, YgdB et PpdC en plus faible abondance.Dans cette étude notre objectif était de décrire la structure des PT4 chez EHEC, en tant que modèle de la famille des pili conservés chez les entérobactéries. La structure est indispensable pour décrire le mode d'action de ces organites. Dans cette étude, nous avons abordé la structure de l'EHEC T4P ainsi que les bases moléculaires de leur assemblage. En raison de la nature flexible et non covalente des fibres T4P, nous avons utilisé une approche intégrative incluant des données obtenues par cryo-microscopie électronique (cryo-ME), spectroscopie RMN, modélisation moléculaire et analyses biochimiques pour déterminer la structure des pili PpdD. Les interactions entre les sous-unités de pilines présentes dans la fibre ont été étudiées par mutagenèse dirigée et analyses fonctionnelles. Ces expériences nous ont permis d’identifier les résidus de PpdD essentiels pour l’assemblage des pili.Les T4aP ont été peu étudiés chez les entérobactéries. Bien que tous les gènes nécessaires soient présents chez E. coli, les conditions induisant leur expression restent inconnues. Cependant, les gènes codant pour les PT4 chez E. coli sont co-régulés avec des gènes codant pour la machinerie impliquée dans l’import d'ADN, ce qui suggère leur rôle dans la compétence naturelle. Afin de faciliter l'étude de PT4, nous avons réalisé une reconstitution fonctionnelle de l'EHEC T4PS chez E. coli K-12 non pathogène par l'expression contrôlée des gènes de PT4 clonés dans un opéron artificiel. Cette construction nous a permis de réaliser des analyses comparatives d'assemblage des pili par le système hétérologue (système de sécrétion de type 2 provenant d'une autre entérobactérie, Klebsiella oxytoca) et le système d'assemblage propre aux PT4 chez EHEC. Leur analyse par cryo-ME a montré que les pili assemblés sont identiques, indiquant ainsi que ce sont les pilines majeures qui déterminent la structure et la symétrie des fibres. Les résultats des études comparatives ont également apporté des informations importantes sur l'assemblage des pili. Nous avons identifié des composants de la machinerie d'assemblage qui interagissent avec la piline majeure PpdD. La formation des dimères de PpdD et ses interactions spécifiques avec les facteurs d’assemblage semblent importantes pour la stabilité de la piline avant l'assemblage en pilus. L’ensemble de nos résultats consitutent des bases pour de futures analyses structurales et fonctionnelles des pili chez les entérobactéries<br>Many bacterial species display surface fibers to interact with the surrounding environment. Type 4 pili (T4P) are long and thin, flexible fibers involved in a variety of functions including adherence, motility, secretion, DNA uptake and biofilm formation. They are composed of thousands of copies of major pilin subunits and are assembled by a protein complex localized in the bacterial envelope. In this study we focused on the T4P from Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7. EHEC is a human foodborne pathogen that causes outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS), which can lead to a lethal outcome. EHEC T4P is composed of the major subunit PpdD and presumably in lower abundance by minor pilins PpdA, PpdB, YgdB and PpdC.This study has aimed to describe the structure of the T4P from EHEC, a representative of pili conserved in enterobacteria. Structural information provides insights that can relate to the mode of action of these organelles. In this project we addressed the structure of the EHEC T4P and the molecular basis of their assembly. Due to the flexible and non-covalent nature of T4P fibers we used an integrative approach to combine information obtained by cryo-electron microscopy, NMR spectroscopy, molecular modeling and biochemical analyses to obtain a high quality structure of the EHEC T4P. In addition, from functional, interaction and mutational analysis we gained insights into the molecular interactions between the pilin subunits present in the fiber. The T4P are poorly studied in enterobacterial systems; despite the presence of all the necessary genes, the conditions that trigger their expression in E. coli are unknown. In addition, in E. coli the T4P-related genes are coregulated with genes encoding the DNA uptake machinery, suggesting their role in natural competence. In order to facilitate the study of T4P, we achieved a functional reconstitution of the EHEC T4PS in the non-pathogenic E. coli K-12 through the controlled expression of the T4P genes cloned together as a single artificial operon. With this accomplishment we were able to perform comparative analyses between pili assembled by a heterologous system (the Type 2 secretion system from another enterobacterium, Klebsiella oxytoca) and by the cognate EHEC T4P assembly system. CryoEM analysis showed that these pili are identical, indicating that major pilins are the key determinants of fiber structure and symmetry. The results also led us to obtain important insights into pilus assembly and to characterize PpdD interactions with the assembly machinery. These interactions and PpdD dimerization were required for pilin stability prior to pilus assembly, highlighting important early steps involving targeting of subunits to the assembly machinery. Together, these results lay the foundations for future structural and functional studies of enterobacterial T4P
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Seow, Vui Yin. "DNA Transformation and Type IV Pili in Neisseria gonorrhoeae." Electronic Thesis or Diss., Sorbonne université, 2024. http://www.theses.fr/2024SORUS049.
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La résistance aux antibiotiques, évidente chez des souches telles que Neisseria gonorrhoeae, est une crise sanitaire mondiale. Il est vital de comprendre son mécanisme, en particulier la transformation naturelle par les Pili de type IV de ces bactéries. Bien qu'elle ait été découverte en 1928, la transformation naturelle n'a toujours pas été élucidée. La dépendance de Neisseria gonorrhoeae à l'égard des Pili de type IV en fait un modèle idéal pour l'étude de ce processus.Cette thèse se lance dans une exploration intensive des processus complexes de transformation de l'ADN au sein de Neisseria gonorrhoeae, répondant ainsi à l'urgence de ce problème critique. Le rôle central de cette étude approfondie est attribué aux pili de type IV, un acteur multifonctionnel et essentiel dans l'orchestration de la transformation de l'ADN.Cette recherche pose méticuleusement les bases, en introduisant des techniques de biologie moléculaire essentielles pour le génie génétique chez N. gonorrhoeae. Nous explorons le développement d'outils, en particulier l'optimisation des milieux et les outils de microscopie adaptés à l'étude de l'absorption et de la transformation de l'ADN. En outre, nos recherches ont mis en évidence une corrélation intrigante entre l'amidon et les acides gras, qui a un impact significatif sur la croissance des gonocoques. Pour comprendre la dynamique des pili de type IV pendant l'absorption de l'ADN, nous avons mis au point des outils et un flux de travail rationalisé capables de visualiser et de quantifier à la fois les pili et les molécules d'ADN. En outre, nous avons automatisé l'analyse des micropiliers d'hydrogel afin d'étudier les propriétés mécaniques des rétractions des pili. En outre, des adaptations des revêtements des micropiliers nous ont permis d'étudier les rétractions des pili en interaction avec l'ADN.Cette étude approfondie comprend également l'examen du comportement des mutants ΔPilV, ΔPilC, et ΔPilD, dévoilant ainsi de profondes connaissances sur les mécanismes de régulation des pili de type IV et leur impact sur la dynamique de l'absorption de l'ADN. Il est particulièrement intéressant de noter que les mutations ΔPilV induisent des altérations dans la translocation de PilE, ce qui entraîne l'émergence de pili plus courts mais efficaces. Cette découverte souligne la nature adaptative de N. gonorrhoeae dans la manipulation de la diversité des pili de type IV pour optimiser les processus d'absorption de l'ADN, une révélation qui revêt une importance considérable dans la lutte contre la résistance aux antibiotiques.En outre, nos études sur d'autres pilines mineures mettent en lumière les altérations du phénotype sans entraver le mécanisme de rétraction des pili. Nos études sur les mutants PilV et PilD mettent en évidence l'influence des modifications post-traductionnelles sur PilE, accentuant ainsi la composition hétérogène des pili de type IV et leur fonctionnalité robuste en tant que polymère.Nous incluons également une courte étude examinant l'interaction entre les espèces de Neisseria commensales et pathogènes dans le contexte de l'absorption de l'ADN, ce qui élargit le champ des implications, invitant à une enquête plus approfondie et élargissant les horizons de ce domaine captivant.Bien que cette expédition laisse certaines questions sans réponse, sa profondeur et son ampleur permettent de mieux comprendre les mécanismes complexes de transformation de l'ADN et le rôle dynamique joué par les pili de type IV dans la remarquable adaptabilité de Neisseria gonorrhoeae. Non seulement cette thèse apporte des solutions aux questions existantes, mais elle ouvre également de nouvelles voies pour la recherche future, suscitant la curiosité et la fascination pour l'élucidation des complexités fonctionnelles des structures pili et leurs implications profondes dans la transformation de l'ADN<br>Antibiotic resistance, evident in strains like Neisseria gonorrhoeae, is a global health crisis. Understanding its mechanism, particularly natural transformation through Type IV Pili in these bacteria, remains vital. Despite discovery in 1928, the specifics of natural transformation remain elusive. Neisseria gonorrhoeae's dependence on Type IV Pili makes it an ideal model for studying this process.This thesis embarks on an intensive exploration into the intricate processes of DNA transformation within Neisseria gonorrhoeae, responding to the urgency of this critical concern. Central to this comprehensive study is the pivotal role attributed to Type IV Pili, a multifunctional and essential player in orchestrating DNA transformation.This research meticulously lays the groundwork, introducing molecular biology techniques essential for genetic engineering within N. gonorrhoeae. We explores tool development, particularly in medium optimization and microscopy tools tailored to study DNA uptake and transformation. Moreover, our investigations uncovered an intriguing correlation between starch and fatty acid, significantly impacting gonococcal growth. To understand Type IV pili dynamics during DNA uptake, we engineered tools and a streamlined workflow capable of visualizing and quantifying both pili and DNA molecules. Additionally, we automated the analysis of hydrogel micropillars to delve into the mechanical properties of pili retractions. Furthermore, adaptations to the micropillar coatings enabled us to study pili retractions interacting with DNA.This in-depth investigation also involves scrutinizing the behaviour of ΔPilV, ΔPilC, and ΔPilD mutants, unveiling profound insights into the regulatory mechanisms of Type IV Pili and their consequential impact on the dynamics of DNA uptake. Particularly noteworthy is the revelation that ΔPilV mutations induce alterations in PilE translocation, resulting in the emergence of shorter yet efficient pili. This discovery underscores the adaptive nature of N. gonorrhoeae in manipulating the diversity of Type IV Pili to optimize DNA uptake processes, a revelation that holds immense significance in combating antibiotic resistance.Furthermore, our studies on other minor pilins sheds light on phenotype alterations without impeding the mechanics of pili retraction. Our studies on PilV and PilD mutants, highlight the influence of posttranslational modifications on PilE, thereby accentuating the heterogeneous composition of Type IV Pili and their robust functionality as a polymer.We also include a short study examining the interplay between commensal and pathogenic Neisseria species within the context of DNA uptake broadens the scope of implications, inviting further inquiry and expanding the horizons of this captivating field.While this expedition leaves certain questions unanswered, its depth and breadth offer extensive insights into the intricate mechanisms of DNA transformation and the dynamic role played by Type IV Pili in the remarkable adaptability of Neisseria gonorrhoeae. Not only does this thesis provide solutions to existing queries, but it also illuminates novel pathways for future research, sparking curiosity and fascination in unravelling the functional intricacies of pili structures and their profound implications in DNA transformation
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Alteri, Christopher. "Novel Pili of Mycobacterium tuberculosis." Diss., Tucson, Arizona : University of Arizona, 2005. http://etd.library.arizona.edu/etd/GetFileServlet?file=file:///data1/pdf/etd/azu%5Fetd%5F1276%5F1%5Fm.pdf&type=application/pdf.
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Couchman, Edward. "Investigating the Type IV pili of Clostridium difficile and Clostridium sordellii." Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/48055.
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Type IV pili (T4P) are the only type of bacterial pili known to be produced by both Gram-negative and Gram-positive organisms. Though the main pilus shaft consists primarily of only one protein (the major pilin), T4P are unusual in their complexity, requiring multiple (10 or more) different protein components for assembly. Like most types of pili, T4P often function as virulence factors. In particular, T4P frequently operate as adhesins, enabling bacteria on which they are present to stick to each other (to form a biofilm or suchlike) or to adhere directly to host cells. Many T4P systems are able to retract, in which case the T4P may mediate flagella-independent motility. Most research into T4P has historically been performed on Gram-negative organisms, with T4P-encoding genes only being identified in Gram-positive organisms more recently. In particular, all sequenced species of the genus Clostridium are known to encode T4P, but only minimal investigation of these systems has been performed to date. In this study, the T4P of Clostridium difficile were investigated. C. difficile is an important pathogen, being the leading cause of antibiotic-associated diarrhoea in the developed world and thus a considerable burden on Western healthcare systems. By investigating the T4P of this species it was hoped to further elucidate its mechanisms of pathogenicity. Data is presented demonstrating the control of T4P expression by cyclic-di-GMP, and identifying which genes are essential for T4P production in C. difficile. Additionally, a genomic analysis of the related pathogen Clostridium sordellii was performed, using the first high quality genome sequence produced for this species. Genes encoding T4P were identified, analysed and investigated. Furthermore, plasmids carrying the genes encoding the species’ key virulence factors (Lethal Toxin, TcsL, and in some cases haemorrhagic toxin, TcsH) were identified. These plasmids appear to be unstable, a fact with significant implications for diagnosis of C. sordellii disease.
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Hendrick, William Anthony. "Molecular Analysis of Type IV Pilus Assembly in Clostridium perfringens." Diss., Virginia Tech, 2016. http://hdl.handle.net/10919/81696.
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Clostridium perfringens is a Gram-positive anaerobe capable of causing disease in humans and many animals. C. perfringens is able to move across surfaces in a manner that is dependent on growth and type IV pili.
Type IV pili are filaments that can be extended away from the cell by rapid polymerization, and retracted by depolymerization. Furthering the understanding of the initial and final energetic states of the pilins will reveal insights into possible mechanisms of type IV pilus assembly. Toward that end, a pilin was purified from the Gram-negative pathogen Pseudomonas aeruginosa and incorporated into an artificial membrane. The pilin was probed by a solid state nuclear magnetic resonance (ssNMR) technique that can determine the angle and depth of insertion of a helical peptide, as well as fluorescent and electron microscopy.
All type IV pilus systems involve the action of an assembly ATPase to provide energy to polymerize the pilus. One proposed mechanism involves two primary proteins: an ATPase and an integral membrane core protein (IMCP). Other type IV pilus proteins are thought to play supportive roles in aiding the traversal of the cell envelope. In order to evaluate this model, the assembly ATPase PilB2 and IMCP PilC2 from C. perfringens were purified and examined for interactions. The evidence presented here suggest that PilB2 and PilC2 do not interact directly, and cannot function as a core assembly apparatus.
The carbonic anhydrase (Cpb) from C. perfringens strain 13 was characterized both biochemically and physiologically. Cpb belongs to the type I subclass of the β class and is the first β class enzyme investigated from a strictly anaerobic bacteria. Kinetic analyses revealed a two-step, pingpong, zinc-hydroxide mechanism of catalysis. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semi-defined medium and an atmosphere without CO₂. The grew well in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases.<br>Ph. D.
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Amerighi, Fulvia. "Impact of S.pneumoniae type-I pilus and its subunits on bacterial adherence to human epithelial cells." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3422591.
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S. pneumoniae is a human pathogen able to cause both invasive and non-invasive diseases as well as to colonize the nasopharyngeal tract of children and adults. Despite pneumococcus is a leading cause of morbidity and mortality worldwide, the pathogenic mechanisms exploited by S. pneumoniae are not yet clear. A critical step is colonization of the nasopharynx and the initial interaction of pneumococci with host cells. Recently, pili were discovered in gram-positive bacteria, mediating critical host-bacterial interactions, such as adherence to the epithelium, biofilm formation and translocation of host mucosal barriers. Thirty to 50% S. pneumoniae strains harbor pilus islet-1 (PI-1) coding for a surface exposed pilus reported to be important both for colonization and virulence. In particular it has been clearly demonstrated that pilus component RrgA is the major determinant for adhesion to epithelial cells in vitro whereas RrgB is important to allow the formation of the polymeric structure into which the adhesin is incorporated.
The data reported in this work contribute to better define the involvement of S. pneumoniae pili in adhesion to human epithelial cells and providing evidence that anti-pilus antibodies are able to prevent bacterial adherence.
In order to test the adhesive attitude of S.pneumoniae strains, we selected a panel of epithelial cell lines differing for their anatomical origin and the capacity to constitute polarized monolayers. Me180 cervical epithelial cells, characterized to form weak adherence junctions and thus expose basolateral surface, result the best model to study pneumococcal adhesiveness. This evidence led us to hypothesize that the pilus ligands may reside in some of the extracellular matrix components rather than in cell associated receptors. Once identified the most appropriate cellular model we started the characterization of pilus dependent adhesion by using TIGR4 mutants lacking pilus components or subpopulations derived from the wild type strain and differing only in pilus expression (highly or poorly piliated). The results reveal that piliated bacteria were drastically more adherent to cells compared to non piliated strains. However, both the lack of the pilus adhesion moiety (RrgA) or of the pilus backbone (RrgB) determined a dramatic reduction of adhesion, suggesting that both the presence of the adhesin and its correct spacing from the bacterial cell were equally important. In addiction we tested the pilus mediated adhesiveness of different pneumococcal strains that could be successfully divided into sub-populations highly (H) or poorly (L) expressing pili. By comparing the H sub-populations we noticed striking difference in their capacity to adhere to host cells. Conventional and immune electron microscopy studies reveal that the capsule thickness is inversely related with the bacterial ability to adhere to host cells likely due to the different exposure of pili on the bacterial surface. Moreover the deletion of the capsular locus in the poorly adherent strain 19FTaiwan14 results in a considerable increase in the adhesion to epithelial cells, at levels comparable to that of poorly capsulated TIGR4 strain. Additionally, in this study, we demonstrate that anti-pilus antibodies were able to significantly impair pneumoccal adhesion to human epithelial cells in strains prominently extruding pili from the capsule. Of interest, we found a monoclonal antibody against RrgA that was able to compete with adhesion in a similar manner with respect to the polyclonal serum against the whole protein. Epitope mapping experiments resulted in the identification of the possible binding region on the RrgA molecule, located in the c-terminal domain. At the moment we are trying to confirm this result by producing point-mutated forms of RrgA with the scope to select mutants that are not recognized by the functional monoclonal antibody and finally complement rrga ko strain with these sequences to confirm the importance of the epitope in the adhesion to human epithelial cells.<br>Streptococcus pneumoniae è un batterio Gram-positivo che fa parte della normale flora microbica che colonizza in modo asintomatico le vie respiratorie. Tuttavia questo microorganismo è anche uno dei principali patogeni umani, può, infatti, causare gravi infezioni del tratto respiratorio sia in forma non invasiva quali otite media, sinusite e polmonite che in casi più gravi forme invasive quali setticemia e meningite. Sebbene S. pneumoniae sia una delle principali cause di mortalità e morbilità nel mondo, i meccanismi patogenetici di questo batterio non sono ancora stati completamente chiariti. Un punto chiave è la colonizzazione del tratto nasofaringeo e l’interazione dei batteri con le cellule ospiti. A questo proposito, recentemente, sono state identificate nei batteri Gram-positivi delle strutture, note come pili, che svolgono un ruolo cruciale nell’interazione ospite-patogeno, sono infatti coinvolti in processi quali: adesione alle cellule epiteliali, formazione di biofilm e traslocazione degli epiteli. Una percentuale variabile tra il 30% e il 50% dei ceppi di S.pneumoniae contiene nel proprio DNA genomico un elemento genetico noto come pilus islet-1 (PI-1) che codifica per una struttura fibrillare, il pilo di tipo1, coinvolto nei processi di colonizzazione e virulenza. In dettaglio, è stato dimostrato che la sub-unità RrgA è coinvolta nell’adesione in vitro dei batteri alle cellule epiteliali mentre la sub-unità RrgB è il principale costituente della struttura del pilo, all’interno della quale è incorporata l’adesina.
I dati riportati in questo lavoro contribuiscono a chiarire il ruolo svolto dal pilo nel meccanismo di adesione di Streptococcus pneumoniae alle cellule epiteliali e forniscono evidenze dell’attività degli anticorpi contro le componenti del pilo di inibire l’adesione dei batteri alle cellule ospiti.
Al fine di valutare le capacità di adesione di Streptococcus pneumoniae, abbiamo selezionato una serie di linee cellulari provenienti da diversi distretti anatomici e con diversa capacità di formare un monostrato di cellule polarizzate in vitro. Tra le linee cellulari testate, il miglior modello per lo studio dell’adesione sono le ME180 (cellule epiteliali di cervice uterina) caratterizzate dal formare giunzioni lasse e quindi consentire l’esposizione di componenti della superficie basolaterale. Questo risultato ci fa ipotizzare che il ligando dei pili possa essere un elemento della matrice extracellulare o un recettore posto sulla superficie basolaterale delle cellule. Una volta identificato il modello cellulare ideale, abbiamo analizzato le capacità di adesione pilo-dipendenti di mutanti che mancano delle componenti del pilo e di sottopopolazioni isolate dal ceppo wild type che differiscono tra loro unicamente per una diversa espressione del pilo, una popolazione è altamente piliata, l’altra scarsamente piliata. I risultati mostrano che la popolazione piliata ha una capacità di aderire alle cellule molto più elevata rispetto alla popolazione non piliata. Inoltre abbiamo osservato che sia l’assenza dell’adesina (RrgA) che del backbone (RrgB) determinano una drastica riduzione dell’adesione sottolineando l’importanza per un corretto funzionamento del pilo sia della presenza dell’adesina che della sua localizzazione nella struttura del pilo. Successivamente abbiamo analizzato il contributo del pilo nell’adesione in diversi ceppi di Streptococcus pneumoniae selezionati sulla base della possibilità di poter isolare le due sottopopolazioni con diversa espressione del pilo (popolazione piliata e non piliata). Prendendo in esame le sottopopolazioni pilate dei ceppi selezionati abbiamo osservato notevoli differenze nella capacità di aderire alle cellule epiteliali. Per spiegare questo fenomeno abbiamo condotto studi di microscopia elettronica convenzionale e immuno-elettro microscopia che hanno evidenziato la presenza di una correlazione inversa tra lo spessore della capsula e le capacità adesive pilo-dipendenti, molto probabilmente dovuta alla diversa esposizione dei pili sulla superficie del batterio. Infatti, la delezione dell’intero locus capsulare in un ceppo che mostra scarsa capacità di adesione alle cellule epiteliali come il ceppo 19FTaiwan14, comporta un notevole aumento dell’adesione a livelli paragonabili al ceppo TIGR4 che è scarsamente capsulato. In questo lavoro abbiamo anche dimostrato che anticorpi prodotti contro le componenti del pilo sono in grado di inibire l’adesione dei batteri alle cellule epiteliali in ceppi in cui il pilo è molto esposto al di fuori della capsula e abbiamo identificato un anticorpo monoclonale anti-RrgA in grado di bloccare l’adesione dei batteri alle cellule ospiti in modo comparabile al siero policlonale prodotto contro l’intera proteina. Studi di epitope mapping hanno portato all’identificazione della regione di RrgA probabilmente coinvolta nel binding con l’anticorpo monoclonale, localizzata nel dominio c-terminale della proteina. Attualmente stiamo cercando di confermare questi risultati inserendo nella regione di RrgA che abbiamo identificato delle mutazioni puntiformi per ottenere forme mutate dell’epitopo che non vengano più riconosciute dal monoclonale e infine complementare il ceppo mutante rrga con queste sequenze per confermare l’importanza di questo epitopo nell’adesione alle cellule epiteliali umane.
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Hartman, Andrea H. "Use of an Inducible Promoter to Characterize Type IV Pili Homologues in Clostridium perfringens." Thesis, Virginia Tech, 2012. http://hdl.handle.net/10919/76874.
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Researchers of <i>Clostridium perfringens</i>, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when <i>C. perfringens</i> was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins.<br>Master of Science
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Paranjpye, Rohinee. "The role of a Vibrio vulnificus type IV pilin in pathogenesis and in persistence in oysters /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/5372.
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Gault, Joseph. "Development of Top-Down Mass Spectrometry Approaches for the Analysis of Type IV Pili." Palaiseau, Ecole polytechnique, 2013. https://tel.archives-ouvertes.fr/pastel-00987029/document.
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La caractérisation de protéines par spectrométrie de masse "top-down" (TDMS) possède plusieurs avantages sur l'approche "bottom-up" (BU), notamment pour la caractérisation des protéoformes; c'est-à-dire l'ensemble des protéines exprimées par la cellule, y compris celles portant les modifications post-traductionnelles (MPT). Dans cette thèse la TDMS a été développée pour l'analyse des pili de type IV (T4P) à la fois sur Orbitrap et sur FT-ICR. Les T4P sont des organelles extracellulaires composées principalement d'une protéine, la piline majeure, qui peut être fortement décorée par des MPT. Pour la piline majeure PilE, du pathogène humain Neisseria meningitidis (Nm), la TDMS a été optimisée pour obtenir la première caractérisation de toutes les protéoformes de PilE exprimées par une souche de référence et un rôle biologique a été proposé pour la MPT glycérophosphate. De plus la TDMS a été appliquée pour une étude à plus grande échelle des MPT des pili provenant des souches cliniques de Nm non-caractérisées. La comparaison des méthodologies TD et BU a révélé à la fois leur complémentarité et la faiblesse inhérente à l'approche BU pour la caractérisation complète de protéoformes. En combinaison avec d'autres techniques structurales, il a été montré que les pili exprimés par les souches de Nm de classe II sont fortement glycosylés, que la glycosylation est dirigée par la séquence de PilE, et que ces sucres modifient fortement la surface des fibres de pili. Ces observations ont amené à une hypothèse nouvelle sur le mécanisme d'évasion immunologique des T4P de classe II chez Nm. De plus la première caractérisation d'une T4P exprimée par une bactérie Gram positif a été réalisée<br>Top-down mass spectrometry (TDMS) is an alternative protein characterisation strategy to the more widespread bottom-up (BU) approach. TDMS has the unique ability to fully characterise the variety of protein products expressed by the cell (proteoforms), including those bearing posttranslational modifications (PTMs). In this thesis TDMS has been developed on both FT-ICR and Orbitrap mass spectrometers for the analysis of type IV pili (T4P). This includes the first T4P to be visualised in a Gram positive bacterium (Streptococcus pneumoniae). T4P are filamentous, extracellular organelles primarily composed of a single protein subunit or major pilin that can be highly posttranslationally modified. For the major pilin, PilE, of the human pathogen Neisseria meningitidis (Nm), TDMS was extensively optimised and the first complete characterisation of all proteoforms of PilE from a single Nm strain performed. A biological role has been proposed for the enigmatic phosphoglycerol PTM. The approach was extended and applied in the first large scale study of PTMs on PilE from uncharacterised, pathogenic strains of Nm. Comparison of the TD and BU methodologies revealed both their complementarity and the inherent weakness of the BU approach for full proteoform characterisation. TDMS was combined with other structural techniques to reveal that pilins from the previously unstudied class II isolates of Nm are extensively glycosylated and that glycosylation is both driven by the primary structure of PilE and has a profound effect on pilus surface topology. These observations have been used to offer the first explanation of how T4P expressed by class II isolates of Nm avoid immune detection
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Lee, Ka Man. "Regulation of expression of the type IV B pili-encoding operon of salmonella typhi /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20LEE.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.<br>Includes bibliographical references (leaves 110-127). Also available in electronic version. Access restricted to campus users.
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Kennouche, Paul. "New insights into meningococcal pathogenesis : exploring the role of the major pilin PilE in the functions of type IV pili Mechanisms of meningococcal type IV pili multiple functions revealed by deep mutational scanning." Thesis, Sorbonne Paris Cité, 2018. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=1972&f=12515.
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Les pili de type IV (PT4) sont des filaments micrométriques qui exercent de multiples fonctions à la surface de nombreux procaryotes. Chez Neisseria meningitidis, les PT4 sont des homopolymères de la piline majeure PilE. Leur implication dans l'agrégation interbactérienne et l'adhésion aux cellules humaines les rend centraux dans la virulence du méningocoque. Cependant, les mécanismes permettant aux PT4 d'exercer ces diverses fonctions restent trop élusifs. Durant ce doctorat, nous avons simultanément déterminé les régions de PilE impliquées dans l'assemblage des pili, l'auto-agrégation, l'adhésion aux cellules humaines et la compétence à la transformation en utilisant la technique de deep mutational scanning. L'analyse approfondie de cette carte fonctionnelle de la séquence de la piline offre de nouvelles perspectives sur les mécanismes de fonctionnement des PT4 : tout d'abord, le domaine hyperconservé 1 de PilE est impliqué dans la régulation de la balance entre la longueur et le nombre des pili ; par ailleurs, nous avons identifié un groupe d'acides aminés électropositifs autour de la lysine 140 requis pour l'agrégation ; enfin, nous montrons l'importance de l'extrémité distale des PT4 dans l'adhésion. En résumé, ces résultats sont en faveur d'un rôle direct de PilE dans l'agrégation et l'adhésion bactérienne et identifient les domaines spécifiquement impliqués dans ces fonctions. Ces travaux ouvrent aussi de nouvelles perspectives sur la pathogénicité de Neisseria meningitidis et pourraient participer au développement de nouvelles thérapies pour combattre les pathologies provoquées par le méningocoque<br>Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are homopolymers of the major pilin PilE. They are crucial for virulence as they mediate interbacterial aggregation and adhesion to host cells although the mechanisms behind these functions remain unclear. During this doctoral work, we simultaneously determined the regions of PilE involved in pili display, auto-aggregation and adhesion to human cells by using deep mutational scanning. Mining of this extensive functional map of the pilin sequence provides new mechanistic insights: first, the hyperconserved 1-domain of PilE was found to be involved in the balance between pili length and number; moreover, we identified an electropositive cluster of residues centered around Lysine 140 necessary for aggregation; finally, we show the importance of the tip of TFP in adhesion. Overall, these results support a direct role of PilE in aggregation and adhesion to host cells and identify these specific functional domains. This doctoral work opens up new perspectives on the pathogenicity mechanisms of Neisseria meningitidis and could help design new therapies to fight meningococcal disease
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Imhaus, Anne-Flore. "Rôle et mode d’action des pilines mineures des pili de type IV de Neisseria meningitidis." Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05T019/document.
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Les pili de type IV (PT4), certainement les organelles les plus répandues des bactéries à Gram-négatif, sont des machineries à multiples fonctions qui jouent un rôle crucial dans la pathogenèse de nombreux pathogènes humains, notamment notre modèle Neisseria meningitidis. L’assemblage des PT4 nécessite une machinerie complexe incluant au moins vingt protéines localisées dans la membrane interne, le périplasme et la membrane externe. Certaines de ces protéines ne sont pas nécessaires pour la biosynthèse des PT4, mais supportent les fonctions qui leur sont associées. Ces protéines, appelées pilines mineures, sont au nombre de trois. Par l’analyse phénotypique des mutants dans les gènes codant pour les pilines mineures, le rôle de chacune a pu être déterminée. Ainsi la piline mineure ComP est nécessaire pour la compétence pour la transformation d’ADN, PilV est requise pour la déformation de la membrane plasmique de la cellule hôte et PilX est essentielle pour l’adhésion des bactéries sur les cellules épithéliales et endothéliales, la formation d’agrégats bactériens et la déformation de la membrane plasmique de la cellule hôte. De nombreuses similarités avec la piline majoritaire laissent penser que les pilines mineures s’insèrent dans la fibre des PT4 pour exercer leurs fonctions, bien que ceci n’a jamais été démontré. Si on connait bien les fonctions des pilines mineures, leur mode d’action n’est toujours pas compris. L’objectif global de ce travail de thèse a été de comprendre comment une fibre protéique peut assurer une diversité de fonctions aussi importante. Pour y parvenir, l’étude du mode d’action des pilines mineures a été entreprise. Contrairement à ce qui prévalait dans le modèle dominant, les pilines mineures PilV et PilX exercent leur fonction à partir de l’espace périplasmique pour moduler la quantité de pili exprimés en surface. En effet, les mutants pilV et pilX présentent respectivement des défauts de piliation de l’ordre de 39% et de 63% par rapport à la souche sauvage. Ces défauts expliquent cependant les phénotypes des mutants. En effet, l’ensemble des fonctions dépendantes des PT4 nécessite une forte quantité de PT4, soit au moins 40% pour l’agrégation et l’adhésion et 70% pour le déclenchement de la réponse cellulaire. Ces résultats révèlent que les pilines mineures sont impliquées dans la biogenèse des PT4 plutôt que dans le support biochimique direct de leurs propriétés. Le défaut de piliation de ces mutants est restauré par l’absence de rétraction, indiquant que les pilines mineures PilV et PilX jouent un rôle dans la stabilité des PT4. Nous avons également montré que la piline mineure ComP est nécessaire pour la piliation et qu’elle présente une fonction redondante avec la piline mineure PilV. Afin de comprendre comment les pilines mineures PilV et PilX exercent leur rôle sur la quantité de pili exprimés en surface, nous avons réalisé une étude structure/fonction de ces deux protéines. Nous avons observé une absence de piliation, en bloquant les pilines PilV et PilX dans la membrane interne, indiquant une interaction directe avec la machinerie des PT4 probablement via la piline majeure PilE. Nous avons également montré qu’il existe une interaction entre les pilines mineures et PilE au niveau de la membrane interne et en amont de l’assemblage des pili. Ces résultats, obtenus par une technique de pontage disulfure, ont cependant besoin d’être confirmés par des contrôles supplémentaires. Par une stratégie de mutagenèse, nous avons enfin mis en évidence que la région D de PilV et les boucles α/β et β2/β3 de PilX sont nécessaires à leur fonctionnement. Ces travaux ont permis de montrer que la quantité de pili exprimés par la bactérie est un facteur déterminant pour définir les propriétés des PT4. Les pilines mineures agissent au niveau du périplasme pour promouvoir la biosynthèse des pili, ce qui met en avant le rôle direct de la piline majeure PilE dans les fonctions associées aux PT4<br>Type IV Pili (TFP) are widespread filamentous organelles extending from the surface of many Gram-negative bacteria that mediate multiple functions and play a key role in the pathogenesis of several important human pathogens, including our model, Neisseria meningitidis. The assembly of TFP requires a complex machinery composed by at least twenty proteins that are localized in the inner membrane, the outer membrane and the periplasm. Three of these proteins, called minor pilins, are not required for the biosynthesis of the TFP, but support their functions. Based on the phenotypes associated with the mutants, their role on TFP functions has been determined. The minor pilin Comp is required for natural competence for DNA transformation, PilV is required for the deformation of the host cell plasma membrane and PilX is essential for the adhesion of bacteria to epithelial and endothelial cells, the bacterial aggregation and the deformation of the host cell plasma membrane. Many similarities with the major pilin PilE suggests that minor pilin are inserted into the fiber of TFP to exert their functions, although it has never been demonstrated. How these proteins carry out their functions mechanistically is not elucidated. The general objective of this thesis was to understand how a single fiber can provide such a variety of functions. To achieve this, the study of the mode of action of minor pilins was undertaken. Contrarily to what has been previously proposed, the PilV and PilX minor pilins seem to exert their functions from the periplasmic space to modulate the amount of surface exposed pili. Indeed, pilV and pilX strains show piliation defects of 39 % and 63 % respectively compared to the wild type. Besides, we have shown that TFP functions require a large amount of TFP, at least 40 % for the aggregation and adhesion and 70% to induce the reorganization of the plasma membrane. Thus these modest decreases in the amount of pili explain the phenotypes of these mutants. These results indicate that the minor pilins are involved in the biogenesis of TFP rather than in the direct support of their biochemical properties. Moreover, the piliation defect of these mutants is restored in the absence of retraction, indicating that the PilV and PilX minor pilins play a role in the stability of TFP. To understand how PilV and PilX minor pilins modulate surface exposed pili level, we performed a structure/ function analysis of these two proteins. Blocking the PilV and PilX minor pilins in the inner membrane abolishes piliation, indicating a direct interaction with the machinery of TFP, probably via the major pilin PilE. We have also shown that an interaction between the minor pilins and the major pilin occurs in the inner membrane and upstream of the pilus assembly. However, these results, obtained by biochemical techniques, need to be confirmed by additional controls. By a mutagenesis strategy, we finally demonstrated that the D region of PilV and the α/β and β2/β3 loops of PilX are necessary for their functions. This study has shown that a relatively modest decrease in the amount of pili displayed on the bacterial surface leads to a strong effect on the functions carried by TFP. Minor pilins act in the periplasm to promote the biosynthesis of pili, which highlights the direct role of the major pilin in the TFP-dependent functions
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Salomonsson, Emelie. "The role of the Type IV pili system in the virulence of Francisella tularensis." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-1656.
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Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia.
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Brown, Daniel Robert. "Systematic analysis in Neisseria meningitidis of proteins that fine-tune functions mediated by type IV pili." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/6834.
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Type IV pili (Tfp) are widespread virulence factors whose multifunctional ability sets them apart from other pili. Tfp mediate adhesion as well as aggregation, competence for DNA transformation and twitching motility (a non-flagellar form of locomotion) in many bacterial pathogens. The molecular mechanisms of these functions and the biogenesis of these pili are not yet fully understood. To better understand Tfp biology, our group started a systematic characterization of all of the proteins involved in Tfp biology in a well-defined genetic background, Neisseria meningitidis strain 8013. Screening of a large library of mutants in this strain followed by an in-depth mining of the genome resulted in the discovery of 23 genes that may be involved in Tfp biology. Seven of these genes encode proteins deemed “accessory” for Tfp biogenesis as the corresponding mutants are piliated. Since previous analysis of one these genes (pilX) led to the finding that it modulates one Tfp-linked function (aggregation), the first aim of my project was to phenotypically characterize mutants in the remaining genes along with double mutants in which filament retraction is abolished by a concurrent mutation in the pilT gene, and strains overexpressing the corresponding proteins with regards to levels of piliation, adhesion, aggregation and DNA competence. This was achieved using a battery of quantitative and qualitative methods well-established in our research group. Results from this study showed that each of the seven proteins plays a role in fine-tuning of Tfp-linked function(s). This completed our systematic study of mutants in N. meningitidis Tfp biology, and gave us a complete picture of the roles of these proteins in Tfp function. This provides us with a platform for further in-depth study of these proteins. In parallel, we attempted to further characterize one of these proteins, PilZ, by determining its localization and protein structure, however, we have been unsuccessful in both efforts so far. Finally we attempted to improve upon the current pilus purification method using a His-tagged version of the major pilus subunit PilE to purify fibres by affinity chromatography. This could be a very useful tool for future studies.
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Murray, Samantha Rose. "Characterization of Type IV Pilus System Genes and Their Regulation in Clostridium perfringens." Thesis, Virginia Tech, 2017. http://hdl.handle.net/10919/86173.
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Clostridium perfringens is a Gram-positive (Gr+) anaerobic pathogen that was found to contain Type IV pilus (T4P) system genes within the genomes of all its sequenced strains. T4P are widely used in Gram-negative organisms for aggregation, biofilm formation, adherence, and DNA uptake. Because few examples of T4P-utilizing Gram-positive bacteria are studied to date, we wanted to characterize the T4P system in this Gr+ bacterium.
To understand the regulation of T4P genes and therefore better understand their expression, we employed the highly powerful next-generation sequencing tool RNA-seq in a variety of conditions. RNA-seq uncovered previously unknown regulatory mechanisms surrounding T4P genes as well as provided transcriptional information for most of the genes in the C. perfringens strain 13 genome. We also utilized reporter gene assays to look at post-transcriptional regulation of T4P promoters.
The wealth of RNA-seq data acted as a jumping-off point for many smaller projects involving transcriptional regulators that may influence T4P expression. We investigated a novel small RNA in close proximity to the major T4P operon, as well as two little-characterized transcriptional regulators that function in the same conditions as T4P genes. RNA-seq also provided data to develop a method for protein purification from C. perfringens without induction.<br>Master of Science
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Nikraftar, Sarah. "Localization of Type IV Pilin Polymerization Proteins in Clostridium perfringens." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/71742.
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Clostridium perfringens is a spore-forming anaerobic Gram-positive rod which has gliding motility through type IV Pili (TFP). Since the discovery of TFP in Gram-positive bacteria is relatively new, more studies are required to understand the mechanism and interaction of the proteins of this machinery. Moreover, the similarities between TFP and type 2 secretion system (T2SS) suggest that C. perfringens has also a T2SS.
We studied the localization of TFP ATPases, PilB1, PilB2 and PilT in Bacillus subtilis to compare the localization in an organism other than C. perfringens and which lacks any known genes similar to TFP. Unlike the case in C. perfringens, PilB1 in B. subtilis localized to the poles in the absence of PilT, with some central foci at the future division sites. Colocalization of PilB1 was also studied with PilT and the results suggested that PilB1 needs PilT to migrate from the poles to the center. Localization of PilB2 in B. subtilis, was similar to the results in C. perfringens and to the localization of PilB1 in B. subtilis. We have not been able to co-express PilB2 with PilT yet. Succeeding in this study will help us better understand the interactions between PilB proteins and PilT.
In another project, we studied a von Willebrand factor Type A-Domain Containing protein (vWA) which is secreted from C. perfringens strain 13. We overexpressed and purified this protein and tested the effects on mammalian cells. We found that the vWA is probably not a toxin but since it seems to bind to macrophage membranes, we propose that the vWA could be part of a toxin complex, probably the subunit of the complex that binds to the host cells.<br>Master of Science
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Friedrich, Carmen [Verfasser], and Lotte [Akademischer Betreuer] Sogaard-Andersen. "Deciphering the assembly pathway of type IV pili in Myxococcus xanthus / Carmen Friedrich. Betreuer: Lotte Sogaard-Andersen." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1045729701/34.
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Rakotoarivonina, Harivony. "Le système d'adhésion de ruminococcus albus : implication de pili de type IV et de deux glycosl-hydrolases." Lyon 1, 2003. http://www.theses.fr/2003LYO10211.
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Ruminococcus albus est une des bactéries cellulolytiques majeures du rumen. L'adhésion de cette bactérie à la cellulose est une première étape indispensable dans le processus de dégradation des fibres végétales. L'objectif de ce travail est d'élucider les mécanismes impliqués dans l'adhésion de R. Albus à la cellulose. Ce travail a permis de caractériser une glycoprotéine de 25 kDa (GP25) sous-produite par un mutant non adhérent de la souche 20 de R. Albus. GP25 est la sous-unité majeure des pili de type IV observés à la surface de R. Albus 20. Ces pili sont nécessaires à l'adhésion de la bactérie à la cellulose. En aval du gène gp25, deux gènes pilA2 et secD-F codant une deuxième protéine homologue aux pilines de type IV et des protéines impliquées dans la sécrétion, sont identifiées. Deux protéines affines pour la cellulose (CBP1 et CBP2) sous-produites par le mutant ont également été caractérisées par électrophorèse bidimensionnelle et spectrométrie de masse (MALDI-TOF). Ces protéines homologues à deux endoglucanases (Cel9E et Cel48A) de R. Albus 8 seraient impliquées dans l'adhésion. Ainsi, le système d'adhésion de R. Albus est multi-factoriel et ferait intervenir des pili de type IV et des glycosyl-hydrolases
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PUJOL, CELINE. "Les pili de type iv chez neisseria meningitidis, elements cles des echanges d'information entre bacteries et cellules eucaryotes." Paris 6, 1999. http://www.theses.fr/1999PA066417.
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Neisseria meningitidis est un pathogene extracellulaire exclusivement humain responsable de septicemies et de meningites. Ce tropisme meninge est specifique de l'infection meningococcique. Les pili de type iv, appendices filamenteux repartis a la surface des bacteries, jouent un role central dans cette interaction. En l'absence de pili, le meningocoque est incapable d'adherer aux cellules de l'hote et de franchir la barriere hemato-encephalique. Dans le but de preciser les mecanismes en cause, nous avons etudie l'interaction du meningocoque avec une monocouche de cellules epitheliales humaines reproduisant les fonctions de barriere qui existent in situ. Deux etapes ont ainsi ete mises en evidence. La premiere necessite la presence des pili et correspond a une adhesion localisee caracterisee par la formation de microcolonies a la surface des cellules epitheliales. La seconde est determinee par une dispersion des bacteries a la surface des cellules, une perte de la piliation, et une adhesion intime des meningocoques a la membrane cellulaire avec parfois formation de piedestals sous-jacents a l'adhesion des bacteries. La survenue de cette seconde etape dite d'adhesion diffuse requiert la premiere phase d'adhesion mediee par les pili et la presence d'une molecule cytoplasmique pilt supposee jouer un role dans la retraction du pilus. Ainsi, sans cette proteine, les meningocoques restent engages dans une etape d'adhesion localisee. Il semble donc qu'un signal percu par les bacteries et capte par les pili, necessitant la presence de pilt, permette l'induction du phenotype dit d'adhesion diffuse. Les pili de type iv du meningocoque pourraient alors etre consideres comme des organes sensoriels et permettraient la perception par la bacterie de signaux cellulaires.
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Lövkvist, Lena. "Receptor Interactions Between Pathogenic Bacteria and Host Cells." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7782.
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<p>This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of <i>Neisseria gonorrhoeae, N. meningitidis</i> and <i>Streptococcus pyogenes</i> on host epithelial cells and <i>in vivo</i>. </p><p><i>N. gonorrhoeae</i> cause the sexually transmitted disease gonorrhoea and <i>N. meningitidis</i> is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, <i>N. meningitidis</i> generally induced an earlier response compared to <i>N. gonorrhoeae</i>, probably as a result of the meningococcal capsule. The role of <i>N. meningitidis</i> serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. </p><p><i>S. pyogenes</i> may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. <i>S. pyogenes</i> ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of <i>S. pyogenes</i> is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of <i>S. pyogenes</i>.</p>
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Harding, Christian Michael. "Discovery and demonstration of functional type IV pili production and post-translational modification by a medically relevant Acinetobacter species." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1428405412.
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Mouville, Clémence. "Interaction entre les pili de type IV et le phage filamenteux MDA : impact potentiel sur la virulence de Neisseria meningitidis." Electronic Thesis or Diss., Université Paris Cité, 2024. http://www.theses.fr/2024UNIP5235.
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Neisseria meningitidis (Nm) est une bactérie commensale du nasopharynx humain qui traverse quelques fois la barrière nasopharyngée et se propage dans la circulation sanguine jusqu'à atteindre les méninges. Un bactériophage filamenteux appelé MDA (Meningococcal Disease Associated) est associé aux infections invasives à méningocoques chez les jeunes adultes. Le MDA semble augmenter l'incidence de la maladie en augmentant la colonisation bactérienne au point d'entrée. L'objectif de ce travail a été de comprendre le mécanisme moléculaire précis de l'infection de Nm par le MDA. L'étude des mutants des gènes impliqués dans la machinerie des pili de type IV (PT4) a montré que l'entrée du phage nécessite un PT4 rétractable. Ce résultat est cohérent avec la littérature sur les phages Ff ou CTX qui interagissent directement avec l'extrémité du pilus. Mais aucune preuve de l'interaction du MDA avec l'extrémité du pilus n'a été trouvée. L'interaction possible entre la fibre du pilus et la capside du phage a été examinée. Comme PilE, la piline majeure, est sujette à variation antigénique, des variants de PilE qui diminuent l'entrée du phage ont été identifiés. L'infection par le phage se produit dans des populations de bactéries exprimant des séquences de PilE spécifiques. Par imagerie, nous avons montré que les pili et les MDA s'associaient ensemble. Une analyse de la charge des acides aminés de PilE et celle de la capside soutient l'hypothèse d'une interaction variable selon le variant de PilE. Finalement, il a été montré que les P4T avec un potentiel électrostatique positif favorisaient l'infection par le phage et permettaient, de plus, une forte adhésion des Nm aux cellules humaines. L'inverse est observé pour les P4T chargés négativement. Ce travail présente également les premières caractérisations de la machinerie de sécrétion du phage. Des études préliminaires avaient montré que dans un biofilm formé sur cellules épithéliales, Nm produisaient soit des pili, soit des phages, mais pas les deux en même temps. Nous avons montré que la sécrétion du phage nécessitait PilQ, PilW et TsaP de la machinerie des P4T. Les analyses bio-informatiques suggèrent que l'ORF8 du phage, qui aurait une activité ATPase, s'associerait avec l'ORF11 pour former un complexe qui interagirait avec la sécrétine bactérienne PilQ. Ce modèle est soutenu par des tests d'interaction par double-hybride. Cette interaction mobiliserait les PilQ de la bactérie, les rendant inaccessible à la machinerie de piliation. La surexpression d'ORF8 entraine une inhibition de la piliation. Ces données permettent d'établir un nouveau modèle d'interaction entre les phages filamenteux et les P4T, ce qui pourrait participer à la sélection des souches pathogènes de Nm<br>Neisseria meningitidis (Nm) is a commensal bacterium of the human nasopharynx that sometimes crosses the nasopharyngeal barrier and spreads through the bloodstream to reach the meninges. A filamentous bacteriophage called MDA (Meningococcal Disease Associated) is associated with invasive meningococcal disease in young adults. MDA appears to increase the incidence of the disease by increasing bacterial colonization at the point of entry. The aim of this work was to understand the precise molecular mechanism of infection of Nm by MDA. The study of mutants of genes involved in the type IV pili (T4P) machinery showed that phage entry requires a retractable T4P. This result is consistent with the literature on Ff or CTX phages, which interact directly with the pilus tip. However, no evidence was found for MDA interacting with the T4P tip. The possible interaction between the pilus fiber and the phage capsid was investigated. Since PilE, the major pilin, is subject to antigenic variation, variants of PilE that reduce phage entry were identified. Phage infection occurs in populations of bacteria that express specific PilE sequences. Using imaging, we showed that pili and MDA associate with each other. An analysis of the amino acid charge of the pilin and that of the capsid supports the hypothesis of a variable interaction depending on the PilE variant. Finally, it was shown that T4P with a positive electrostatic potential favored phage infection and allowed the bacteria to adhere strongly to human cells. The opposite is observed for negatively charged T4P. This work also presents the first characterizations of the phage secretion machinery. Previous studies had shown that in a biofilm formed on epithelial cells, Nm produce either pili or phages, but not both at the same time. We showed that phage secretion requires PilQ, PilW and TsaP of the pili machinery. Bioinformatic analyses suggest that phage ORF8, which has ATPase activity, associates with ORF11 to form a complex that interacts with the bacterial secretin PilQ. This model is supported by two-hybrid interaction assays. This interaction would mobilize the bacterial PilQ, making them inaccessible to the piliation machinery. Overexpression of ORF8 inhibits piliation. These data provide a new model for the interaction between filamentous phages and T4P, which could be involved in the selection of pathogenic strains of Nm
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Santos, Moreno Javier. "Molecular mechanism of pseudopilus assembly in the Klebsiella oxytoca type II secretion system." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC216/document.
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Le système de sécrétion de type II (SST2) permet la sécrétion de protéines repliées à travers la membrane externe chez les bactéries à Gram-négatif. Le SST2 est une nano-machine enchâssée dans l’enveloppe bactérienne, proche par sa composition et structure aux systèmes d’assemblage des pili de type IV (PT4) impliqués, entre autres, dans d’adhésion et motilité. Chez Klebsiella oxytoca, la surexpression des gènes pul codant le SST2 permet l’assemblage de pili composées des sous-unités PulG. Ceci suggère qu’en conditions physiologiques l’assemblage d’un pseudopilus périplasmique permet la sécrétion du substrat spécifique du SST2, la pullulanase. Dans ce projet nous avons exploré le mécanisme moléculaire de l’assemblage du pseudopilus en se focalisant sur les interactions de PulG avec les composants du SST2 dans la membrane interne. En utilisant l’approche de double-hybride bactérien, nous avons établi le réseau d’interactions de PulG avec les pseudopilins mineures PulH, I, J et K et avec la plateforme d’assemblage (PA). Pour valider ces interactions, nous avons combiné des techniques de biochimie (co-purification par affinité, pontage cystéine et chimique) avec des analyses fonctionnelles de sécrétion et de formation du pseudopilus. Nous avons mis en évidence des interactions entre PulG et les protéines de la PA, PulF et PulM, et nous avons analysé en détail l’interface PulG-PulM. Les résultats suggèrent la formation d’un complexe PulK-I-J-H-G dans la membrane interne impliqué dans des étapes précoces de la formation du pseudopilus, à travers les interactions de PulG et PulH avec PulM et PulF. Nos données expérimentales suggèrent un rôle majeur de PulM dans la sécrétion, vraisemblablement durant l’assemblage du SST2 et l’élongation du pseudopilus. Nos travaux collaboratifs mettant en jeu l'analyse par spectroscopie de masse et en dynamique moléculaire in silico révèlent le rôle essentiel des résidus conservés Glu5 et Thr2 de PulG, requis pour l’interaction avec PulM. Ces données suggèrent que Glu5 participe à l'extraction de PulG de la membrane, en neutralisant la charge positive de son peptide N-terminal par des interactions intramoleculaires. Ces résultats permettent d'établir un modèle détaillant les étapes initiales de l’assemblage des pseudopili dans la membrane interne, relevant pour de futures études sur le SST2 et nanomachines homologues. sécrétion de protéinespili de type 4 assemblage de fibres complexes protéiques membranairesinteractions protéine-protéinemicroscopie à immuno-fluorescence simulations en dynamique moléculairedouble-hybride bactérien spectrométrie de masse nanomachines bacteriennes<br>The type II secretion system (T2SS) drives the translocation of folded, periplasmic proteins across the outer membrane in Gram-negative bacteria. Secretion is carried out by an envelope-spanning nanomachine that is similar to the apparatus that builds type IV pili (T4P), bacterial surface filaments involved in adhesion, motility and other functions. In the Pul T2SS of Klebsiella oxytoca, overexpression of pul genes in plate-grown bacteria allows the assembly of T4P-like surface fibres made of PulG subunits, suggesting that a periplasmic pseudopilus fibre plays a role in the secretion of the type II substrate pullulanase under physiological conditions. In this project, we explored the molecular mechanism of pseudopilus assembly by focusing on the interaction between PulG and the T2SS inner membrane and pseudopili components. The network of interactions of PulG with the minor pseudopilins PulH, I, J and K and the assembly platform (AP) components was established using bacterial two-hybrid analysis. To validate these interactions, we combined biochemical approaches (affinity co-purification, chemical or cysteine cross-linking) with functional assays of secretion and pseudopilus formation. We provide evidence of the interaction between PulG and the AP proteins PulF and PulM, and delve into the PulG-PulM interface. Our results point to the formation of a PulK-I-J-H-G complex in the plasma membrane involved in early steps of fibre assembly, with a determinant role for PulG and PulH interaction with PulM and PulF. We obtained experimental evidence supporting a major role for PulM in pseudopilus assembly and protein secretion, probably by intervening in the assembly of the T2SS apparatus and in pseudopilus elongation. The results of experimental and in silico studies in collaboration with experts in mass spectrometry and molecular dynamics support the essential role of the highly conserved PulG residues Glu5 and Thr2, which participate in PulM binding. In addition, Glu5 probably favours PulG membrane extraction by neutralising its N-terminal positive charge through intra-molecular interaction. These findings shed new light on early membrane events during fibre assembly, and open new and exciting avenues in research on T2SSs and related nanomachines.protein secretiontype 4 pilifibre assemblymembrane protein complexprotein-protein interactionsimmunofluorescence microscopymolecular dynamics simulationsbacterial two-hybrid assaymass spectrometrybacterial nanomachines
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Bordeleau, Éric. "Régulation du c-di-GMP et rôle de ce messager secondaire dans la formation de pili de type IV chez Clostridium difficile." Thèse, Université de Sherbrooke, 2014. http://hdl.handle.net/11143/5385.
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Malgré la découverte du c-di-GMP en 1987, ce n’est que durant la dernière décennie que l’importance de ce messager secondaire dans la régulation des phénotypes bactériens a été exposée. Synthétisé par des diguanylate cyclases (DGC) et dégradé par des phosphodiestérases spécifiques (PDE), le c-di-GMP est prédit pour être un messager secondaire très répandu chez les bactéries et pratiquement exclusif à celles-ci. Le c-di-GMP est particulièrement reconnu pour son rôle dans la transition des bactéries motiles et planctoniques vers la formation de biofilm chez les bactéries à Gram négatif telles qu’Escherichia coli, Pseudomonas aeruginosa et Vibrio cholerae. De plus, le c-di-GMP est impliqué dans la régulation de l’expression de certains facteurs de virulence chez certaines bactéries. Ainsi, il est possible de révéler les mécanismes de régulation de certains phénotypes importants par l’étude de la signalisation à c-di-GMP dans une bactérie donnée. Clostridium difficile est une bactérie pathogène causant des diarrhées nosocomiales, des colites et pouvant causer des décès chez l’Homme. Les phénotypes impliqués dans la pathogenèse de C. difficile et leur régulation demeurent en grande partie méconnus. Le génome de C. difficile 630 était prédit être capable de coder pour 37 DGC et PDE putatives, un nombre en apparence élevé pour une bactérie à Gram positif. L’objectif global de mon doctorat était de déterminer si la signalisation à c-di-GMP était fonctionnelle chez C. difficile puis de déterminer le rôle du c-di-GMP chez cette bactérie. Dans un premier projet, mes travaux de doctorat ont permis de démontrer que la majorité des 37 DCG et PDE putatives chez C. difficile 630 sont fonctionnelles. Les 31 DCG et PDE les plus conservées dans les différentes souches de C. difficile ont été exprimées dans V. cholerae afin d’évaluer indirectement leur capacité de synthèse et de dégradation du c-di-GMP en mesurant leur impact sur motilité et la formation de biofilm de V. cholerae. La surexpression d’une DGC chez V. cholerae réduit la motilité par flagelle et augmente la formation de biofilm, alors que l’inverse est observé lors de la surexpression d’une PDE. De plus, l’activité d’une DCG, CD1420 (renommée DccA, CD630_14200), et une PDE, CD0757 (renommée CD630_07570) a été démontrée plus explicitement par des essais enzymatiques in vitro. Ainsi, ce projet a exposé l’important potentiel de la signalisation à c-di-GMP chez C. difficile, jusqu’alors étudiée presque exclusivement chez les bactéries à Gram négatif. Dans un deuxième projet, mes travaux de doctorat ont permis de démontrer le rôle des pili de type IV (T4P) dans l’agrégation de C. difficile et la régulation de leur expression par un riborégulateur à c-di-GMP. Les riborégulateurs sont des structures ARN situées dans la région 5’UTR des gènes capables de réguler l’expression des gènes en aval en fonction de la liaison d’un métabolite spécifique. Parmi les 16 riborégulateurs à c-di-GMP prédits dans le génome de C. difficile 630, le riborégulateur c-di-GMP-II Cdi2_4 est situé en amont du locus principal de synthèse de T4P. Mes travaux ont permis de montrer que l’augmentation de la concentration de c-di-GMP intracellulaire se traduit par une augmentation de l’expression des gènes de T4P, la formation de T4P à la surface des cellules et l’agrégation dépendante des T4P. De plus, le mécanisme de régulation du riborégulateur Cdi2_4 a été démontré in vitro. La liaison du c-di-GMP au riborégulateur Cdi2_4 prévient la formation d’un terminateur transcriptionnel et favorise ainsi la transcription des gènes de T4P en aval. Depuis la mise en évidence de la signalisation à c-di-GMP chez C. difficile dans la première partie de mon doctorat, un certain nombre de phénotypes régulés par c-di-GMP chez cette bactérie ont pu être déterminés ou prédits. Notamment, le c-di-GMP inhibe la transcription des gènes des flagelles en se liant au riborégulateur c-di-GMP-I Cd1 en amont et inhibe indirectement la production des toxines TcdA et TcdB. La démonstration de l’effet positif du c-di-GMP sur l’agrégation des cellules via les T4P, dans la deuxième partie de mon doctorat, contribue à notre compréhension de la signalisation à c-di-GMP chez C. difficile. Il apparait que le c-di-GMP inhibe la motilité et favorise la formation de structures pluricellulaires chez C. difficile à l’instar de plusieurs bactéries, néanmoins par des mécanismes de régulation distincts.
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Potapova, Anna [Verfasser], and Lotte [Akademischer Betreuer] Soegaard-Andersen. "Regulation of type IV pili formation and function by the small GTPase MglA in Myxococcus xanthus / Anna Potapova ; Betreuer: Lotte Soegaard-Andersen." Marburg : Philipps-Universität Marburg, 2020. http://d-nb.info/1214368220/34.
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Soyer, Magali. "Mécanismes moléculaires de la colonisation de l’endothélium par Neisseria meningitidis." Thesis, Paris 5, 2012. http://www.theses.fr/2012PA05T080.
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Les infections bactériennes touchant la circulation sanguine conduisent à un vaste éventail de graves pathologies, comme les chocs septiques ou les infections locales (endocardites et méningites). Neisseria meningitidis colonise avec succès l’endothélium vasculaire et cause des sepsis sévères. Ces infections résultent de la colonisation des cellules endothéliales de l’hôte, étape clef de la pathophysiologie à laquelle les travaux présentés dans ce manuscrit se sont intéressés. La colonisation de l’endothélium par N. meningitidis est un processus complexe qui implique l’adhésion et la multiplication des bactéries à la surface des cellules endothéliales dans le contexte particulier de la circulation sanguine, où des forces mécaniques sont générées par le flux sanguin sur les objets circulants. Bien que de nombreuses études se soient intéressées à l’interaction entre les cellules endothéliales et N. meningitidis, plusieurs aspects demeurent incertains comme par exemple l’impact des contraintes générées par le flux sanguin et la participation relative des deux partenaires de l’interaction dans la colonisation de l’endothélium par N. meningitidis.L’adhésion de la bactérie à la surface des cellules endothéliales est dépendante de facteurs bactériens (les pili de type IV, PT4) et induit une réponse de la part de la cellule hôte, qui se traduit par un remodelage de la membrane plasmique et une réorganisation du cytosquelette d’actine sous les microcolonies. Dans un premier temps, ces travaux de thèse montrent que la réponse cellulaire induite par N. meningitidis participe activement à la colonisation. En effet, la formation de projections membranaires permet à chaque bactérie de la microcolonie d’établir des contacts avec la cellule hôte, nécessaires à la résistance des microcolonies face aux forces mécaniques générées par le flux sanguin. De plus, nous montrons que la protéine PilV, composant des PT4, est impliquée dans le remaniement de la membrane plasmique et la réorganisation du cytosquelette. Nous avons développé une méthode combinant vidéo-microscopie et analyse de fluorescence pour décrypter les événements précoces prenant place lors du contact entre les bactéries et la surface des cellules hôtes. Nous avons alors montré que le remodelage de la membrane induit par N. meningitidis ne dépend pas de la réorganisation du cytosquelette d’actine au site d’infection mais plutôt des propriétés intrinsèques de la bicouche lipidique.Dans un second temps, nous nous sommes intéressés aux étapes tardives de l’infection, c'est-à-dire à l’initiation d’un nouveau cycle de colonisation. Bien que solidement ancrées à la surface des cellules par l’intermédiaire des projections membranaires, quelques bactéries se détachent des microcolonies pour coloniser des nouveaux sites au sein de l’hôte. Nous avons démontré l’importance de modifications post-traductionnelles de la piline majeure dans cette étape de l’infection et caractérisé les mécanismes impliqués.Cette étude a permis d’affiner les mécanismes impliqués dans l’induction de la réponse cellulaire induite par N. meningitidis et son impact sur la colonisation efficace de l’endothélium par ce pathogène<br>Bacterial infections targeting the bloodstream lead to a wide array of severe clinical manifestations, such as septic shock or focal infections (endocarditis and meningitis). Neisseria meningitidis colonizes successfully the vascular wall and causes severe sepsis. Such infections result from an efficient colonization of host endothelial cells, a key step in meningococcal diseases which has been the subject of the work presented here. Endothelium colonization by N. meningitidis is a complex process implying bacterial adhesion and multiplication on the endothelial cell surface in the specific context of the bloodstream, where mechanical forces generated by the blood flow are applied on circulating bacteria. Even though many studies focused on the interaction between N. meningitidis and the endothelial cell, many aspects remain elusive, such as the impact of shear stress generated drag forces and the relative contribution of the two partners involved in this interaction.Adhesion to the endothelial cell surface is dependent on bacterial factors called type IV pili (Tfp) and leads to induction of a host cell response, characterized by a local remodeling of the plasma membrane and reorganization of actin cytoskeleton underneath bacterial microcolonies. First, we have shown that the cellular response induced by N. meningitidis actively participate in the colonization process. Indeed, membrane deformation allows contact with every bacterium inside the microcolony, which is necessary for microcolony resistance to mechanical forces. Additionally, we have demonstrated that the PilV protein, a Tfp component, is involved in plasma membrane remodeling and actin cytoskeleton reorganization. We designed a method combining high resolution live-cell fluorescence video-microscopy and fluorescence quantification to decipher the early events induced on contact of bacterial aggregates with the host cell surface. Using this technique we have shown that membrane remodeling does not rely on actin cytoskeleton reorganization but rather on intrinsic properties of the lipid bilayer. Second, we focused on latter steps of the infection process when initiation of a new colonization cycle is initiated. While firmly attached to the host cell surface through the membranous projections, some bacteria can detach from the microcolony to disseminate throughout the host. We have demonstrated the importance of post-translational modification of the major piline in this step and characterized the underlying mechanisms.This work allows refinement of the molecular mechanisms involved in the induction of the cellular response induced by N. meningitidis and its impact on successful endothelium colonization by this pathogen
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Basso, Pauline. "Exolysine, un facteur de virulence majeur de Pseudomonas aeruginosa." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV063/document.
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Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier<br>Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment
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Cheung, Fei Wai. "The effect of bile salts on expression from the pil and rci promoters associated with the type IVB pilus-encoding operon of salmonella enterica serovar typhi /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20CHEUNG.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.<br>Includes bibliographical references (leaves 98-116). Also available in electronic version. Access restricted to campus users.
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Forslund, Anna-Lena. "Identification of new virulence factors in Francisella tularensis." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknat- och Medfak), 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-30857.
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Francisella tularensis, the causative agent of tularemia, is a highly virulent bacterium with an infection dose of less than ten bacteria. The ability of a pathogen to cause infection relies on different virulence mechanisms, but in Francisella tularensis relatively few virulence factors are known. Two F. tularensis subspecies are virulent in humans; the highly virulent subspecies tularensis, also referred to as type A, and the less virulent subspecies holarctica, also called type B. The aim of this thesis has been to improve the knowledge regarding the ability of Francisella to cause disease, with the emphasis on surface located and membrane associated proteins and structures. In addition I have also investigated how virulence is regulated by studying the role of the small RNA chaperone, Hfq. The genome of Francisella appears to encode few regulatory genes. In my work I found that Hfq has an important role in regulation of virulence associated genes in Francisella. Similar to what has been found in other pathogens, Hfq functions in negative regulation, and this is the first time a negative regulation has been described for genes in the Francisella pathogenicity island. Another protein with a key role in virulence is a homologue to a disulphide oxidoreductase, DsbA, which was identified as an outer membrane lipoprotein in Francisella. A dsbA mutant was found to be severely attenuated for virulence and also induced protection against wild-type infections, thus making it a candidate for exploration as a new live vaccine. Additional genes with homology to known virulence determinants include a type IV pilin system. The pilin homologue, PilA, was identified to be required for full virulence in both type A and type B strains. In addition, genes involved in pili assembly and secretion, pilC and pilQ, were also found to be virulence associated in the type A strain. In summary, dsbA, hfq and type IV pili associated genes were indentified to be virulence determinants in F. tularensis. DsbA is a potential target for drug development and a dsbA mutant a candidate for a new live vaccine strain. Furthermore the identification of Hfq as a novel regulatory factor opens new insights into the virulence regulatory network in Francisella.
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Williams, Danielle A. "The AlgZ/R Two-Component System Is Responsible for Attenuation of Virulence in Pseudomonas aeruginosa." Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etd/3340.
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Pseudomonas aeruginosa is an important opportunistic pathogen. Many P. aeruginosa virulence factors are regulated by the AlgZ/R two component system. AlgZ is the sensor histidine kinase which phosphorylates AlgR, the response regulator. AlgR activates transcription of different gene targets based upon its phosphorylation state. The genes that encode AlgZ and AlgR are transcribed in an operon. While regulation of algR expression has been well studied, regulation of algZ expression has not. Using a pilW mutant in concert with algZTF-lacZ transcriptional fusion, we conducted a transposon mutagenesis to identify algZ regulators. We identified an unknown autoregulatory loop. The type IV pilus minor pilins prevent the phosphorylation of AlgR by AlgZ . This inhibition of the AlgZ/R system subsequently down-regulates both the expression of the fimU operon and the algZ/R operon. Because AlgR regulates virulence, it is possible that virulence can also be reduced by targeting activation of the AlgZ/R system.
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Theophilou, Elena Stella. "Development of a novel genetic system for generation of markerless deletions in Clostridium difficile." Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9616.
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C. difficile is an obligate anaerobic, Gram-positive, rodshaped and spore-forming bacterium. It is a well-recognised causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile has emerged as an important nosocomial pathogen in recent years, associated with considerable morbidity, mortality and economic burden. Despite its importance, functional genomic studies have been lagging behind in comparison to other enteric pathogens. This is attributed to the fact that C. difficile is difficult to manipulate genetically and the lack of robust, reproducible mutagenesis systems for many years. The ideal mutation for robust functional genomic studies is a markerless, in-frame deletion of the gene of interest. All systems developed for C. difficile, up to the start of this study, involve insertional inactivation of the gene of interest. This study describes the development of a novel genetic system for C. difficile, to create precise and markerless chromosomal deletions, using the meganuclease ISceI. For validation of the system, the addBA genes in C. difficile were deleted. The AddAB enzyme complex is important in the survival of many bacteria, since it maintains genome integrity, by the repair of double-strand breaks. Deletion of addBA in C. difficile did not significantly affect growth and viability, but the mutant strains were sensitive to DNA damaging agents. In addition, it was shown that C. difficile is capable of initiating the SOS response after DNA damage and that AddAB is not necessary for the induction of this response. The genetic system was further optimised to delete type IV pili (TFP)- associated genes, particularly pilT (CD3505) and pilA (CD3507), to investigate twitching motility. TFP are important in virulence and pathogenesis of many bacteria and twitching motility is often involved. TFP in C. difficile may be expressed in vivo during infection and may be involved in biofilm formation and colonization. To study potential TFP-mediated motility, a non-flagellated C. difficile strain was first constructed by deleting the fliC gene. The pilT gene, predicted to encode a protein involved in TFP retraction, was then deleted in the ΔfliC strain. A ΔpilT strain was also generated. Preliminary experimental work using these strains did not show any evidence for twitching motility and no difference between the ΔpilT strains and the parental strains. Examination of cells from the ΔfliC strain, under various conditions, did not reveal any pili, which indicates that TFP are regulated in C. difficile and that the TFP locus might be repressed at the transcriptional level. Preliminary work to investigate an intergenic region located upstream of the TFP locus in C. difficile, that might be involved in regulation, suggested that transcription is being initiated within a 500 bp region upstream of the CD3513 gene.
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Varga, John Joseph. "The Role of CcpA in Regulating the Carbon-Starvation Response of Clostridium perfringens." Diss., Virginia Tech, 2006. http://hdl.handle.net/10919/29759.
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Clostridium perfringens is a significant human pathogen, causing 250,000 cases of food poisoning in addition to several thousand potentially lethal cases of gas gangrene each year in the United States. Historically, work in this field has centered around toxin production, as C. perfringens can produce over 13 toxins. This work expands the knowledge of the starvation-response of C. perfringens, which includes several potential virulence factors, sporulation, motility and biofilm formation. Sporulation protects cells from a variety of stresses, including starvation. Efficient sporulation requires the transcriptional regulator CcpA, mediator of catabolite repression. Sporulation is repressed by glucose, but, surprisingly, in a CcpA-independent fashion. C. perfringens cells in a biofilm are resistant to a number of environmental stresses, including oxygen and antibiotics. Biofilm formation is repressed by glucose, and other carbohydrates, independently of CcpA. Gliding motility, a type four pili (TFP)-dependent phenomenon, affords C. perfringens with a mechanism for moving across a solid surface in response to carbohydrate starvation, while carbohydrates supplementation at high levels delay the initiation of the motility response. CcpA is required for the proper initiation of motility, a ccpA<sup>-</sup>C. perfringens strain showed a considerable increase in the time to initiation of motility on lactose and galactose, and was unable to move at all in the presence of glucose. Gliding motility represents the most significant finding of this work. TFP were previously undescribed in any Gram-positive bacterial species, and this work produced genetic evidence suggesting their presence in all members of the clostridia, and physical evidence for TFP-dependent gliding motility in a second species, C. beijerinckii.<br>Ph. D.
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Mikaty, Guillain. "Rôle des Pili de type IV dans le réarrangement de la surface cellulaire eucaryote induite par Neisseria meningitidis et conséquences sur la colonisation des barrières cellulaires." Paris 5, 2009. https://hal.archives-ouvertes.fr/tel-01262387.
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Neisseria meningitîdis est une bactérie à Gram négatif, à la fois commensale et pathogène de l'espèce humaine. Au cours de ces travaux, nous nous sommes intéressés à l'interaction entre cette bactérie et son hôte. La colonisation des cellules par N, meningitîdis est un processus complexe qui intègre : l'adhésion aux cellules de l'hôte dépendante de la présence de Pili de type IV (Pt4), la multiplication et la survie des bactéries, la formation de microcolonies et leur maintien sur les cellules puis l'envahissement progressif de la surface cellulaire. Cette étape de colonisation est centrale dans le cycle commensal et dans la pathogenèse de cette bactérie. Elle a donc été largement étudiée par le passé. Cependant deux aspects avaient été négligés dans la plupart de ces études. (1) La colonisation des cellules humaines se fait dans un contexte de flux de liquides, mucus ou sang, qui génère des forces hydrodynamiques qui s'opposent à la colonisation. (2) L'interaction entre une bactérie et une cellule eucaryote implique une participation de chacune des deux cellules. Laicolonisation des cellules humaines par N. Meningitîdis induit plusieurs changements physiologiques dont une réorganisation complexe du cytosquelette d'actine aboutissant à la formation de projections membranaires à la surface des cellules hôte. C'est donc en intégrant ces deux aspects que nous avons abordé la question de la colonisation de l'hôte par N. Meningitidis. Ces travaux de thèse ont permis de montrer que la réorganisation du cytosquelette d'actine induite par la bactérie participait activement à la colonisation. La formation des projections membranaires permet à la plupart des bactéries au sein de la microcolonie, d'établir des liaisons robustes avec la membrane plasmique. Ces liaisons sont les seules qui permettent à N. Meningitidis de résister aux forces hydrodynamiques présentes dans son environnement naturel. De plus, nous avons montré que les Pt4 de la bactérie sont le vecteur moléculaire de l'induction de la réorganisation du cytosquelette. Nous avons identifié deux protéines dans ces pili qui assurent la fonction inductrice. Ces protéines, les pilines mineures PilV et PiLX, sont nécessaires à l'induction de la réorganisation'du cytosquelette d'actine, et par conséquent, à la colonisation des cellules humaines par N. Meningitidis. Dans ces deux piline^ mineures, une région particulière caractérisée par un pont disulfure est essentielle à cette fonction. Cette région est exposée à l'extérieur de la bactérie et pourrait agir comme ligand d'un récepteur cellulaire. Ces travaux ont associés une nouvelle fonction aux Pt4 de la bactérie. De plus, nous avons découvert une fonction inédite' à cette réponse cellulaire dans la colonisation de N. Meningitidis. Le détournement des voies de signalisation de la cellule hôte permet à la bactérie de résister aux conditions hydrodynamiques de son environnement naturel. Mots clefs : Colonisation, N. Meningitidis, Pili de type IV, pilines mineures, Cytosquelette d'actine, réarrangement de la surface cellulaire, Forces hydrodynamiques
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Arantxa, Camus Etchecopar. "Mécanismes moléculaires impliqués dans la formation de biofilm à l’interface eau-composés organiques hydrophobes." Thesis, Pau, 2014. http://www.theses.fr/2014PAUU3032/document.
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Les composés organiques hydrophobes (HOC), une grande famille de molécules naturelles ou d’origine anthropique incluant les lipides et les hydrocarbures, constituent une part significative de la matière organique dans les écosystèmes marins. Du fait de leur faible solubilité dans l’eau, les bactéries qui les dégradent requièrent la mise en place de fonctions cellulaires spécifiques permettant d’augmenter la fraction assimilable de ces HOC. La formation de biofilms à l’interface eau-HOC est une de ces stratégies adaptatives. C’est le cas pour Marinobacter hydrocarbonoclasticus SP17, modèle d’étude utilisé au laboratoire, qui est capable de former des biofilms sur un large spectre de HOC métabolisables tels que les alcanes, les triglycérides et les alcools gras. Le but de mes recherches consistait à améliorer la compréhension du processus d’adhésion et de développement des biofilms sur les HOC, à travers la caractérisation fonctionnelle de 10 gènes candidats mis en évidence lors d’analyses d’expression en protéomique et en transcriptomique. Pour mener à bien ce projet, des outils génétiques et une caractérisation fonctionnelle propre à chaque gène ont dû être développés. L’étude fonctionnelle du gène MARHY2686 a relevé son implication dans la formation de biofilm sur les alcanes. La co-expression de MARHY2686 et des gènes adjacents MARHY2687 et MARHY2685 en transcriptomique, leur distribution phylogénétique et leur conservation de la synthénie suggèreraient que ces trois gènes soient impliqués dans le même processus biologique. D’après l’identité forte de 36 % qui existe entre la protéine MARHY2686 et une protéine périplasmique AdeT d’un système de pompe d’efflux tripartite d’Acinetobacter baumanii, cette protéine, en association avec MARHY2687 et MARHY2685, pourrait faire partie d’un système de ce type. Par ailleurs, des observations ont permis d’envisager une implication potentielle de ce gène dans l’assimilation des HOC ou dans l’accumulation des réserves lipidiques intracellulaires. M. hydrocarbonoclasticus SP17 utilise les pili de type IV lors de la formation de biofilm sur les HOC. Ces appendices interviennent lors de l’adhésion de cette souche à des HOC ainsi que dans un processus de détachement d’un support hydrophobe. Les pili pourraient soit intervenir directement pour permettre à la bactérie de se détacher de la surface à laquelle elle s’est adhérée, soit indirectement par l’action de bactériophages. La présence d’une mobilité de type twitching sur les HOC a pu être également envisagée. Enfin, le rôle du système de sécrétion de type VI (T6SS), connu pour permettre à la bactérie d’interagir avec une cellule hôte, lors de la formation de biofilm mono-spécifique sur HOC, où aucun autre microorganisme que M. hydrocarbonoclasticus SP17 n’est présent, a été étudié<br>Hydrophobic organic compounds (HOC), a large family of naturally-produced or anthropogenic molecules including lipids and hydrocarbons, represent a significant part of organic matter in marine ecosystems. Because of their low solubility in water, bacteria that degrade those compounds require the establishment of specific cell functions to increase their biodisponibility. Biofilm formation in water-HOC interface is one of these adaptations. The model of bacteria used in our laboratory, Marinobacter hydrocarbonoclasticus SP17, is able to form a biofilm on a wide range of HOC, such as alkanes, fatty alcohols and triglycerides, in order to use them as a carbon and energy source. The main purpose of my work was to broaden the knowledge of how bacteria adhere to and from biofilms on HOC, through the functional characterization of 10 candidate genes highlighted during proteomic and transcriptomic studies. Genetic tools and a gene-specific functional characterization have been developed in order to carry out this project. Functional study conducted on MARHY2686 revealed its involvement in the formation of biofilm on alkanes. Co-expression of MARHY2686 and the adjacent genes MARHY2687 and MARHY2685 durnig transcriptomic analysis together with their phylogenetic distribution and synteny conservation suggest that these three genes are involved in the same biological process. According to the high peptide sequence identity between MARHY2686 and AdeT, a periplasmic protein of a tripartite efflux pump system of Acinetobacter baumanii, MARHY2686 in combination with MARHY2687 and MARHY2685 could be the components of such a system. Other phenotypic observations would consider the involvement of MARHY2686 either in the assimilation of HOC or in the accumulation of intracellular lipid reserves. M. hydrocarbonoclasticus SP17 uses type IV pili during biofilm formation on HOC. These appendages are involved in the adhesion of this strain to and in a detachment process from HOC. Type IV pili could either act directly to allow bacteria to detach from the surface to which it is adhered, or indirectly through the action of bacteriophages. The presence of twitching motility on HOC has also been suggested. Finally, the role of the type VI secretion system (T6SS), a well-known protein system which allows interactions between bacteria and host cells, during the formation of a mono-species biofilm on HOC where no other microorganism than M. hydrocarbonoclasticus SP17 is present, has been studied
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Black, Wesley P. "Regulation of Exopolysaccharide Production in Myxococcus Xanthus." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/30250.
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The surface gliding motility of Myxococcus xanthus is required for a multicellular developmental process initiated by unfavorable growth conditions. One form of the M. xanthus surface motility, social (S) gliding, is mediated by the extension and retraction of polarly localized type IV pili (Tfp). Besides Tfp, exopolysaccharides (EPS), another cell surface associated component, are also required for M. xanthus S motility. Previous studies demonstrated that the Dif chemotaxis-like signal transduction pathway is central to the regulation of EPS production in M. xanthus. Specifically, difA, difC and difE mutants were found to be defective in EPS production and S motility. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA, respectively, are therefore positive regulators of EPS. This study, undertaken to better understand the regulation of EPS production, led to a few major findings. First, DifD and DifG, homologous to CheY and CheC, respectively, were found to be negative regulators of EPS production. Both DifD and DifG likely function upstream of the DifE kinase in EPS regulation. DifB, which has no homology to known chemotaxis proteins, was found not to be involved in EPS production. Secondly, this study led to the recognition that Tfp likely function upstream of the Dif pathway in the regulation of EPS production. Extracellular complementation experiments suggest that Tfp may act as sensors instead of signals for the Dif chemotaxis-like pathway. We propose a regulatory feedback loop that couples EPS production with Tfp function through the Dif signaling proteins. Lastly, we sought to identify additional genes involved in EPS production. Our efforts identified a mutation in a separate chemotaxis gene cluster as a suppressor of difA mutations, suggesting potential cross-talks among the multiple chemotaxis-like pathways in M. xanthus. In addition, we identified twenty-five previously uncharacterized genes that are predicted to be involved in M. xanthus EPS production. These genes appear to encode additional EPS regulators and proteins with biosynthetic function.<br>Ph. D.
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Dienst, Dennis. "Untersuchungen zu Funktion und Struktur des Regulatorproteins Hfq in Synechocystis sp. PCC 6803." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16250.
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Das phylogenetisch weit verbreitete RNA-bindende Protein Hfq ist an einer Vielzahl von Prozessen innerhalb des bakteriellen RNA-Metabolismus, insbesondere im Rahmen der post-transkriptionellen Genregulation durch kleine RNAs (sRNAs) beteiligt. Hfq-Proteine zählen zu der Familie der Sm- und Lsm-Proteine und zeichnen sich strukturell durch die funktionelle Ausbildung ringförmiger Homohexamere aus. Cyanobakterielle Orthologe zeigen gegenüber den gut untersuchten Hfq-Proteinen aus E. coli und anderen Proteobakterien eine schwache Sequenzkonservierung und bieten auch daher einen interessanten Ansatzpunkt für die Untersuchung riboregulatorischer Prozesse in diesen Organismen. In der vorliegenden Arbeit werden einleitende Untersuchungen zu Funktion und Struktur des orthologen Hfq-Proteins aus dem einzelligen Modell-Cyanobakterium Synechocystis sp. PCC 6803 vorgestellt. Die Inaktivierung des hfq-Gens (ssr3341) führte in diesem Organismus zum Verlust der phototaktischen Motilität. Mithilfe elektronenmikroskopischer Analysen konnte dieser Phänotyp auf das Fehlen von Typ IV Pili zurückgeführt werden. Microarray-Analysen wiesen in der deltahfq-Mutante für 31 Gene eine veränderte, in den meisten Fällen reduzierte Transkriptakkumulation nach. Am stärksten betroffenen waren Gene bzw. Operone, welche dem Regulon des cAMP-Rezeptorproteins Sycrp1 zugeordnet werden und zum Teil nachweislich an der Motilität von Synechocystis-Zellen beteiligt sind. Weitere vergleichende Expressionsanalysen identifizierten mithilfe eines speziellen Tiling-arrays ferner zwei „intergenisch“ kodierte potenzielle sRNAs, Hpr1 und Hpr3, deren Transkriptmengen signifikant von der hfq-Inaktivierung beeinflusst werden. Kristallstrukturdaten deuten zusammen mit den Ergebnissen aus in vitro-Bindungsstudien und genetischen Komplementierungsexperimenten - trotz starker Konservierung zentraler struktureller Charakteristika - neuartige biochemische und funktionelle Eigenschaften des Hfq-Proteins aus Synechocystis sp. PCC 6803 an. Funktionelle Implikationen werden im strukturellen und phylogenetischen Kontext diskutiert.<br>The phylogenetically conserved RNA binding protein Hfq is a key player in bacterial RNA metabolism, particularly with regard to sRNA-mediated post-transcriptional gene regulation. Hfq proteins belong to the well-conserved family of Sm- and Lsm proteins and are characterized by the formation of homo-hexameric ring-shaped structures. In comparison with well-studied Hfq proteins from E.coli and other proteobacteria the cyanobacterial orthologues show rather poor sequence conservation. Therefore, they provide a quite interesting background for analyzing riboregulatory processes in these organisms. In this work, the orthologous Hfq protein from the unicellular model cyanobacterium Synechocystis sp. PCC 6803 has been initially characterized on the functional and structural level. Insertional inactivation of the hfq gene (ssr3341) led to a non-phototactic phenotype that was due to the loss of type IV pili on the cell surface, as demonstrated by electron microscopy. Microarray analyses revealed a set of 31 genes with altered transcript levels in the knock-out mutant. Among the most strongly affected genes, there were members of two operons that had previously been shown to be involved in motility, controlled by the cAMP receptor protein Sycrp1. Further comparative transcriptional analyses using custom tiling arrays revealed two putative sRNAs (Hpr1 and Hpr3) from intergenic regions, whose transcript levels appeared to be significantly affected by hfq-inactivation. Structural analyses, genetic complementation as well as RNA-binding studies in vitro indicate that the Hfq orthologue from Synechocystis sp. PCC 6803 exhibits novel biochemical and functional properties, though retaining general structural features of its proteobacterial counterparts. Functional implications are discussed with regard to structural und phylogenetic considerations.
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Sundin, Charlotta. "Type III Secretion Mediated Translocation of Effector Exoenzymes by Pseudomonas aeruginosa." Doctoral thesis, Umeå : Univ, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-174.
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Virion, Zoé. "Interaction de Neisseria meningitidis avec les cellules endothéliales humaines : rôle des glycosylations des récepteurs cellulaires eucaryotes Receptor recognition by meningococcal type IV pili relies on a specific triantennary N-glycan Sialic acid‐mediated allosteric activation of β2-adrenoceptors An ADAM-10 dependent EPCR shedding links meningococcal interaction with endothelial cells to purpura fulminans". Thesis, Sorbonne Paris Cité, 2018. https://wo.app.u-paris.fr/cgi-bin/WebObjects/TheseWeb.woa/wa/show?t=2296&f=12491.
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Neisseria meningitidis est une bactérie commensale du rhinopharynx portée de façon asymptomatique par 10 à 35% de la population. Pour une raison encore inconnue à ce jour, cette bactérie est capable de traverser la barrière épithéliale et ainsi proliférer dans la circulation sanguine de l’hôte, où elle est capable d’adhérer aux cellules endothéliales grâce aux pili de type IV. Sur ces cellules, le méningocoque interagit spécifiquement avec le récepteur d’adhésion CD147 et le récepteur β2-adrénergique qui active la signalisation sous la colonie bactérienne adhérente, favorisant l’ancrage de celle-ci à la surface cellulaire et l’ouverture des jonctions cellulaires. Nous avons montré que l’adhésion aux cellules endothéliales humaines est dépendante des motifs spécifiques de N-glycosylation portés par les récepteurs cellulaires eucaryotes. Les résultats montrent que le troisième site de N-glycosylation du récepteur CD147 est indispensable pour permettre l’adhésion de la bactérie, et que l’interaction est permise par les résidus d’acide sialique portés par ces chaînes de glycosylation. Les acides sialiques sont également essentiels pour l’interaction du méningocoque avec le récepteur β2-adrénergique et l’activation de la signalisation cellulaire sous la colonie. Les résultats montrent que la forme Neu5Ac (acide N-acétylneuraminique) des acides sialiques retrouvée dans l’espèce humaine pourrait participer à la spécificité d’espèce de N. meningitidis, la plupart des autres espèces de mammifères présentant une forme Neu5Gc (acide N-glycolylneuraminique). Nous avons ainsi pu montrer qu’une partie de la spécificité d’espèce du méningocoque est liée à l’interaction des pili de type IV avec des sucres spécifiques<br>Neisseria meningitidis is a commensal bacteria found in the nasopharynx of 10 to 35% of the population. For a still unknown reason, the bacteria a able to cross the epithelial barrier and to reach the bloodstream, where it can proliferate and adhere to the human endothelial cells via the type IV pili. The meningococcus specifically interacts with the adhesion receptor CD147 and the β2-adrenergic receptor, responsible for the activation of signaling under the adherent colony. We showed that the adhesion to the human endothelial cells is dependent on specific N-glycosylation patterns carried by the cellular receptors. The results show that the third N-glycosylation site of CD147 is essential to the adhesion of the bacteria, and that the interaction is due to the presence of sialic acid residues of the N-glycosylation chains. The sialic acids are also essential for the interaction of the meningococcus with the β2-adrenergic receptor and the activation of the signaling under the colony. The results show that the sialic acid of the form Neu5Ac (N-acetylneuraminic acid) found in humans could explain de species specificity of the meningococcal infection, most of the other mammals species possessing a Neu5Gc (N-glycolylneuraminic acid) form of the sialic acid. So we showed that a part of the species specificity of the meningococcus is due to the interaction of the type IV pili with specific glycosylations
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Ke-YingHsieh and 謝可盈. "The Contribution of Type IV Pili in Clostridium difficile pathogenesis." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/7w95kp.
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Chiang, Yi Chien, and 江宜蒨. "Characterization of the Type IV pili gene cluster Streptococcus sanguinis SK36." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/88998232805241370382.
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碩士<br>長庚大學<br>生物醫學研究所<br>99<br>Streptococcus sanguinis is a primary colonizer of human tooth and an opportunistic pathogen for subacute endocarditis. A Type IV pili (Tfp) gene cluster was reported in the complete genome of Streptococcus sanguinis SK36 recently. The goal of this research aimed to analyze the expression and function of this gene cluster. The Tfp gene cluster is composed of total 16 genes, from pilB to pilD. A contiguous transcript was detected between pilD and the downstream lytB by RT-PCR, suggesting that lytB is also part of the operon. Three transcription initiation sites, 153- (P1), 536- (P2) and 837-base (P3) 5’ to the translation start site of pilB, respectively, were detected by rapid amplification of cDNA ends (5’ RACE) analysis. Both the P2 and P3 mapped to a σ70-like promoters (5’-TTGACA-N17-TATACT), whereas only an extended -10 sequence was observed with the P1. An anti-PilA antibody was generated and used to examine the structure of the pil cluster encoded products by transmission electron microscopy (TEM). A short hair-like structure was observed in the wild-type SK36 but not the Pil-deficient mutant strain, indicating that pil cluster is responsible for the synthesis of this structure. Furthermore, the pil-deficient mutant strains exhibited reduced biofilm formation. However, neither the adherence to Hela cells nor the twitching motility was affected by the deletion in pil cluster. Taken together, these results suggest that the pil cluster is responsible for the synthesis of a surface structure, and this structure is associated with biofilm formation. The multiple transcription initiation sties with long 5’ untranslated regions suggested the presence of a complex regulation system for the expression of the pil cluster.
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Barnett, Timothy Carew. "A genetic and functional analysis of type IV pili produced by Aeromonas bacteria." Thesis, 1999. https://eprints.utas.edu.au/19091/1/whole_BarnettTimothyCarew1999_thesis.pdf.
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Bacteria belonging to the genus Aeromonas are ubiquitous water-borne organisms
that are also present in many foods. Some strains are human gastrointestinal pathogens.
However, the disease-causing mechanisms of these bacteria are not well understood.
This is particularly true for intestinal colonisation, which is a critical step in the disease
process. When this thesis commenced, there was evidence that filamentous surface
structures (type IV pili) purified from diarrhoea-associated species had an important role
in colonisation. These pili were designated "bundle-forming pili" (Bfp) because of their
tendency to form bundles linking bacteria.
The initial aim of this thesis was to clone the genes encoding Bfp pili from a
diarrhoeal isolate of Aeromonas veronii biovar sobria (strain BC88). Although
traditional approaches (transposon mutagenesis, screening of libraries with degenerate
oligonucleotide probes, DNA probes, and a Bfp antiserum) were unsuccessful at
achieving this, a small region of a gene encoding the Bfp pilin protein was ultimately
cloned using a degenerate PCR approach. Moreover, a second type IV pilus gene cluster
was also cloned from this strain. Characterisation of this newly discovered pilus family
(designated Tap, for "type IV Aeromonas pilus") was then undertaken.
The tap gene cluster from A. veronii biovar sobria BC88 was shown to comprise
four genes that were all transcribed in the same direction. These genes exhibited high
sequence homology to genes encoded by similar gene clusters, identified by other
investigators, from strains of A. hydrophila, Vibrio cholerae and V. vulnificus. To assess the significance of Tap pili for Aeromonas virulence, the distribution of
the tap genes was determined in Aeromonas reference strains (including both
pathogenic and non-pathogenic species), and in a range of clinical and environmental
isolates of those species most commonly associated with human gastrointestinal disease.
The tap cluster was present in all Aeromonas strains tested. A defined mutation in tapA
from A. veronii biovar sobria BC88 was then constructed. Inactivation of this gene did
not alter the ability of this strain to adhere to epithelial or intestinal cells in vitro, or to
colonise the intestinal tract of infant mice (colonisation and competition experiments). Furthermore, the tapA mutant strain was not attenuated in its ability to cause disease in
rabbits (removable intestinal tie adult rabbit diarrhoeal model; performed by Dr. M. J.
Albert, International Center for Diarrheal Disease Research, Dhaka, Bangladesh).
To investigate the expression and assembly of Tap pili, an antiserum was
constructed against a recombinant protein produced by overexpression of tapA in
Escherichia coli. Western blot analysis showed that TapA was expressed. However, Tap
pili on the cell surface were not seen by immune electron microscopy. (Previous studies
in our laboratory have demonstrated that Bfp pill are the predominant structures
expressed on the surface of Aeromonas bacteria during growth in vitro.)
Hence, this thesis identified a second family of type IV pili in Aeromonas species.
The function and expression results obtained to date suggest that Tap pili are not as
significant as Bfp pili for colonisation of the intestinal tract. However, the widespread
distribution of the tap genes in Aeromonas species, as well as in other bacterial
pathogens, suggests that they are important for some aspect of the biology of these
organisms.
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Bulyha, Iryna [Verfasser]. "Regulation of the type IV pili localization in Myxococcus xanthus / vorgelegt von Iryna Bulyha." 2010. http://d-nb.info/1004808496/34.
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Tseng, Tzu Ying, and 曾姿穎. "Characterization and functional analysis of the type IV pili gene cluster in Streptococcus sanguinis SK36." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/33875483902144865967.
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碩士<br>長庚大學<br>生物醫學研究所<br>101<br>Streptococcus sanguinis is a member of the dental plaque and occasionally causes infective endocarditis. Thus far the gene cluster (pil) encoding type IV pili (Tfp) was found only in the genome of Streptococcus sanguinis SK36. Previous studies by using 5’ RACE analysis revealed 3 putative transcription initiation sites 5’ to the pil cluster. Short hair-like structures were observed on the surface of SK36 by using anti-SSA_2315 (PilA) antiserum under transmission electron microscopy. However, the biological functions of the Tfp in S. sanguinis SK36 remains unknown. This study aims to analyze the expression and function of the pil cluster. By using various pil promoter-reporter fusion strains, it was found that all 3 promoters were functional. The activity of a transcriptional fusion containing all 3 promoters was higher in the ccpA-deficient host than that in the wild-type background, indicating that the expression of the pil operon is subject to the regulation of CcpA. Western analysis of the PilA protein indicated that the biogenesis of Tfp was regulated by growth phases, with the highest expression at the early stationary phase. Inactivation of SSA_2313-2315 led to a 40% reduction in adherence to HeLa cells and squamous cell carcinoma (SCC-4) compared to the wild-type strain. Taken together, the expression of the pil cluster was regulated by a complex system and the biosynthesis of Tfp was closely associated with the development of growth phase. The binding of S. sanguinis SK36 Tfp to host cells supports the role of Tfp in the pathogenesis of S. sanguinis SK36.
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Wu, Hui Yu, and 吳蕙妤. "Regulation and functional analysis of the type IV pili gene cluster in Streptococcus sanguinis SK36." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/m9564z.
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Chiang, Poney Che. "Molecular investigation of the role of type 4 pili ATPases involved in twitching motility of Pseudomonas aeruginosa." 2005. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=371008&T=F.
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Wu, Chia Hua, and 吳佳樺. "Functional analysis of the type IV pili gene cluster in the twitching and non-twitching Streptococcus sanguinis strains." Thesis, 2017. http://ndltd.ncl.edu.tw/handle/p57s9h.
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碩士<br>長庚大學<br>生物醫學研究所<br>106<br>A type IV pili (Tfp) gene pil cluster of 16 or 17 genes is found in strains of Streptococcus sanguinis but not in other oral streptococci. Although this cluster is highly conserved among S. sanguinis strains, only a few Tfp-expressing strains exhibit twitching motility. To understand the basis for twitching activity, this study examined the pil cluster and the twitching activity of 40 clinical S. sanguinis isolates. Among all isolates, pil-specific PCR products were observed in 39 isolates, indicating that the pil cluster is present commonly in clinical isolates. Although the pil cluster in the non-twitching type-strain SK36 and the twitching clinical strain #10 differs only in the central portion of the cluster, further analysis with all sequenced S. sanguinis strains failed to draw a clear connection between this region and the twitching phenotype. On the other hand, strain #10 expressed a higher amount of pilT and generated longer Tfp compared to SK36, although both strains shared a common 5’ flanking region of the pil cluster. While inactivation of the expression of either the entire cluster (#10∆Tfp) or the gene encoding the retraction ATPase (#10∆pilT) abolished the twitching activity of S. sanguinis #10, #10∆pilT exhibited a wild-type level of adherence to host epithelial cells. Furthermore, #10∆pilT also formed thicker biofilm compared to wild-type #10 in a batch system. Taken together, Tfp, but not the twitching motility, are crucial for the attachment of S. sanguinis. The Tfp-driven motility, however, may reduce the interaction between bacteria in biofilm formation.
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Tammam, Stephanie. "Characterization of PilP from the Type IV Pilus System of Pseudomonas aeruginosa." Thesis, 2012. http://hdl.handle.net/1807/43398.
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Pathogenic bacteria employ a number of mechanisms to induce infection and survive in host tissues, including toxin secretion and the formation of protective multicellular structures called biofilms. Type IV Pili (T4P) are highly conserved organelles essential for both the establishment of infection and biofilm maturation. The goal of this research is to gain a molecular level understanding of the function of the highly dynamic T4P of Pseudomonas aeruginosa. The pilMNOPQ operon encodes 5 members of a transmembrane complex that facilitates pilus function. While PilQ is the putative outer membrane secretin through which the pilus exits the cell, the roles of the PilM/N/O/P proteins are less well defined. Using both in vivo and in vitro techniques our characterization of PilP has provided significant insight into organization of the apparatus. PilP is an inner membrane lipoprotein essential for T4P function, but lipidation is dispensable, suggesting that its interactions with other T4P components are sufficient for PilP function. We showed that PilN/O/P form a stable heterotrimer when expressed in E. coli, and we suggest that they form a similar subcomplex in P. aeruginosa. Additionally we were able to show that PilP is also able to interact with a periplasmic fragment of the outer membrane pore protein PilQ. Structural and bioinformatics studies suggest that the organization of PilN/O/P/Q complex is similar to that of the transenvelope complex of another important Gram-negative virulence factor – the Type II Secretion System (T2SS). Our structural and functional characterization of PilP, the PilN/O/P complex and the striking similarities between the T4P and T2S systems, as well as important differences that make each molecular machine unique, will be presented.