Escherichia coli est une espèce bactérienne à multiples facettes. En effet, certaines souches sont présentes à l’état commensal au niveau intestinal, ou sont utilisées comme probiotiques. À l’inverse, d’autres souches sont responsables d’infections intestinales ou extra-intestinales chez l’Homme et les animaux à sang chaud. Les souches d’E. coli pathogènes extra-intestinales (ExPEC) sont responsables de nombreuses maladies infectieuses (méningites néo-natales, infections urinaires, septicémies ou infections respiratoires). Plusieurs facteurs de virulence ont été identifiés chez les souches ExPEC (adhésines, invasines…) et notamment la protéine IbeA, mise en évidence dans une souche isolée d’un cas de méningite néo-natale humaine. Le gène ibeA est retrouvé chez différentes souches ExPEC, dont certaines d’origine aviaire. Il est localisé au sein de l’îlot génomique GimA, sur un des quatre opérons de cet îlot, entre ibeR codant un potentiel régulateur et ibeT codant un potentiel antiporteur. La protéine IbeA a été décrite comme jouant un rôle dans l’invasion bactérienne des cellules endothéliales microvasculaires de cerveau humain (HBMEC). Afin de mieux comprendre le rôle d’IbeA dans le processus infectieux et l’invasion cellulaire, nous avons étudié l’implication d’IbeA dans l’adhésion de la souche ExPEC d’origine aviaire BEN2908 puis tenté de déterminer la localisation de cette protéine et son lien avec la protéine IbeT. L’étude phénotypique comparative de la souche BEN2908 et de son mutant ?ibeA nous a montré qu’IbeA intervenait dès le stade de l’adhésion aux HBMEC. Des tests d’adhésion en absence des fimbriae de type 1 (adhésine majeure de notre souche) nous ont montré que dans ce contexte, IbeA n’avait pas d’action sur l’adhésion. Ce résultat nous a suggéré qu’il pouvait y avoir une baisse d’expression des fimbriae de type 1 à la surface bactérienne dans un mutant ?ibeA, ce que nous avons montré par dots blots. Pour comprendre comment IbeA entraînait une modification de l’expression des fimbriae de type 1, nous nous sommes intéressés au contrôle de l’expression des gènes de l’opéron fim. Nous avons ainsi montré que le promoteur de ces gènes, localisé sur un élément invertible, était préférentiellement dans une orientation ne permettant pas la transcription des gènes fim dans un mutant ?ibeA. Nous avons ensuite mis en évidence chez le mutant ?ibeA une baisse de l’expression des gènes fimB et fimE qui codent pour deux recombinases participant au contrôle de l’orientation de l’élément invertible. Ces baisses d’expression de fimB et fimE pourraient expliquer la diminution d’expression des fimbriae de type 1 dans le mutant ?ibeA. Enfin, des phénotypes similaires à ceux du mutant ?ibeA ont été observés chez un mutant ?ibeT. La localisation d’IbeA est indispensable pour comprendre comment cette protéine peut agir sur l’expression des recombinases FimB et FimE. Nous avons localisé IbeA dans le compartiment cytoplasmique, mais l’incertitude sur la fonctionnalité d’IbeA dans les constructions génétiques utilisées nécessite de confirmer ces premiers résultats. Enfin, nous avons recherché un rôle métabolique pour IbeA et IbeT étant données les homologies d’IbeT avec des transporteurs de composés carbonés. Nous avons observé qu’un mutant ?ibeT présentait un retard de croissance par rapport à la souche sauvage et au mutant ?ibeA dans des cultures en milieu minimum avec du fumarate, du succinate, du malate ou de l’aspartate comme seule source de carbone. Ces résultats suggèrent un lien entre le métabolisme de certains dicarboxylates, l’expression des fimbriae de type 1 et les protéines IbeA et IbeT. Ils ouvrent de nombreuses perspectives pour la compréhension du mécanisme d’action d’IbeA et IbeT. / Escherichia coli is bacterial species with multiple facets. Indeed, some strains are present at a commensal state in the intestinal tract of humans and warm-blooded animals, or are used as probiotics. Conversely, other strains are responsible for intestinal or extra-intestinal infections in Humans and warm-blooded animals. Extra-intestinal pathogenic E. coli (ExPEC) strains are responsible for multiple infectious diseases (neonatal meningitis, urinary tract infections, septicaemias or respiratory infections). Several virulence factors have been identified in ExPEC strains (adhesins, invasins,…) and notably the IbeA protein, originally identified in a strain isolated from a case of human neonatal meningitis. The ibeA gene is found in different ExPEC strains, of including strains avian origin. It is located on one of the four operon of the GimA genomic island, between ibeR coding a putative regulator and ibeT coding a putative antiporter. The IbeA protein is known for its role in bacterial invasion of human brain microvascular endothelial cells (HBMEC). In order to better understand the role of IbeA in the infectious process and cellular invasion, we have studied the involvement of IbeA in adhesion of the avian pathogenic E. coli strain BEN2908 and attempted to determine the localisation of this protein and its link with the IbeT protein. The comparative henotypic study of strain BEN2908 and its ?ibeA mutant showed that IbeA was involved in the adhesion to HBMEC. Adhesion tests in the absence of type 1 fimbriae ( the major adhesin of our strain) showed that IbeA did not have a direct role in adhesion in this context. This result suggested there could be a decrease in type 1 fimbriae expression at the bacterial surface in the ?ibeA mutant. This was demonstrated by dot blots. To understand how IbeA led to a modification of type 1 fimbriae, we investigated the role of IbeA in the control of the expression of genes that belong to the fim operon. Thus we showed that the promoter of these genes, located on an invertible element, was preferentially in an orientation preventing transcription of the fim genes in the ?ibeA mutant. Then, we highlighted in the ?ibeA mutant, a decrease of expression of the fimB and fimE genes encoding two recombinases involved in the orientational control of invertible element. These decreases of fimB and fimE expression could explain the reduction of type 1 fimbriae expression in the ?ibeA mutant. Lastly, phenotypes similar to that of the ?ibeA mutant were observed in a ?ibeT mutant. The localisation of IbeA is necessary to understand how this protein can act on fimB and fimE expression. We localised IbeA in the bacterial cytoplasm, but the doubt on the functionality of IbeA in the genetic constructions used demands that these results be confirmed. Finally, we have looked for a metabolic role for IbeA and IbeT, given the IbeT homology with carbon compound transporters. We have observed that, in minimal broth with fumarate, succinate, malate or aspartate as sole carbon sources, the ?ibeT mutant presented a lower growth rate than the wild type strain and ?ibeA mutant. Altogether, these results suggest a link between metabolism of dicarboxylates, type 1 fimbriae expression and IbeA and IbeT proteins. They open numerous perspectives for the comprehension of IbeA and IbeT mechanisms.

Afin de comprendre la biogénèse et l'assemblage des fimbriae de Streptococcus salivarius, les gènes inactivés de deux mutants ayant une production réduite de fimbriae ont été analysés. Chez le mutant C34, le transposon est inséré entre les gènes cysD et ssaO988, codant respectivement pour une O-acetylhomosérine sulfhydrylase et une hydrolase des haloacides déhalogénases. Les analyses de la transcription ont montré que les transcrits ne sont pas perturbés. Chez le mutant DU, le transposon est inséré dans le gène dagA codant pour une D-alanine/glycine/Na perméase et les analyses de la transcription ont montré l'absence du transcrit. La complémentation en trans a été effectuée, mais la méthode d'immunoempreinte n'a pas permis de détecter le retour à une expression normale de fimbriae. L'absence du transporteur de la D-alanine, stéréoisomère retrouvé exclusivement dans la paroi, pourrait perturber l'ancrage des fimbriae par une modification de la composition et/ou de la stabilité de la paroi bactérienne.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 175-194).

Proefschrift Maastricht. / Lit.opg.

Thesis (Ph. D.)--University of Washington, 2002. / Includes bibliographical references (leaves 159-186).

Bacterial resistance to antibiotics is increasing at a high rate in both developing and developed countries. To circumvent the problem of drug-resistant bacterial pathogens, we need to develop new effective methods, substances, and materials that can disarm and prevent them from causing infections. However, to do this we first need to find new possible targets in bacteria to approach and novel strategies to apply.Escherichia coli (E. coli) bacteria is a normal member of the intestinal microflora of humans and mammals, but frequently cause diverse intestinal and external diseases by means of virulence factors, which leads to hundreds of million sick people each year with a high mortality rate. An E. coli bacterial infection starts with adhesion to a host cell using cell surface expressed adhesion polymers, called adhesion pili. Depending on the local environment different types of pili are expressed by the bacteria. For example, bacteria found in the gastrointestinal tract commonly express different pili in comparison to those found in the urinary tract and respiratory tract. These pili, which are vital for bacterial adhesion, thereby serve as a new possible approach in the fight against bacterial infections by targeting and disabling these structures using novel chemicals. However, in order to develop such chemicals, better understanding of these pili is needed.Optical tweezers (OT) can measure and apply forces up to a few hundred pN with sub-pN force resolution and have shown to be an excellent tool for investigating mechanical properties of adhesion pili. It has been found that pili expressed by E. coli have a unique and complex force-extension response that is assumed to be important for the ability of bacteria to initiate and maintain attachment to the host cells. However, their mechanical functions and the advantage of specific mechanical functions, especially in the initial attachment process, have not yet been fully understood.In this work, a detailed description of the pili mechanics and their role during cell adhesion is presented. By using results from optical tweezers force spectroscopy experiments in combination with physical modeling and numerical simulations, we investigated how pili can act as “shock absorbers” through uncoiling and thereby lower the fluid force acting on a bacterium. Our result demonstrate that the dynamic uncoiling capability of the helical part of the adhesion pili modulate the force to fit the optimal lifetime of its adhesin (the protein that binds to the receptor on the host cell), ensuring a high survival probability of the bond.iiiSince the attachment process is in proximity of a surface we also investigated the influence of tether properties and the importance of different surface corrections and additional force components to the Stokes drag force during simulations. The investigation showed that the surface corrections to the Stokes drag force and the Basset force cannot be neglected when simulating survival probability of a bond, since that can overestimate the probability by more than an order of magnitude.Finally, a theoretical and experimental framework for two separate methods was developed. The first method can detect the presence of pili on single cells using optical tweezers. We verified the method using silica microspheres coated with a polymer brush and E. coli bacteria expressing; no pili, P pili, and type 1 pili, respectively. The second method was based on digital holography microscopy. Using the diffraction of semi-transparent object such as red blood cells, we showed that this method can reconstruct the axial position and detect morphological changes of cells.

Neisseria meningitidis is a causative agent of meningitis and septicaemia. Pili are one of the major virulence factors that contribute to the pathogenicity of N. meningitidis. Pili of Neisseria are type IV fimbriae composed primarily of thousands of identical pilin subunits. Pilin of N. meningitidis is post-translationally modified by trisaccharide, phosphorylcholine and -glycerophosphate. The genes involved in pilin expression, pilin glycosylation and phosphorylcholine modification are phase variable (high frequency ON/OFF switching of expression). The function of pilin post-translational modifications and their phase variable expression in host:pathogen interactions is unknown. The phase variable expression of glycosylation in bacteria has been proposed to function in bacterial adherence and immune avoidance. However, the function of pilin glycosylation in N. meningitidis is unclear. Phosphorylcholine is expressed in a number of respiratory organisms including P. aeruginosa (on teichoic acid), S. pneumoniae (on lipoteichoic acid) and H. influenzae (on LPS). Phosphorylcholine in these organisms is important in colonisation of the nasopharynx and invasion of the epithelium. Studies on N. meningitidis pilin post-translational modifications have been restricted by difficulties in purification of pilin protein. In this thesis, we evaluated current pilin purification methods and established an efficient method of purifying pilin from N. meningitidis by Flag-tag purification system. Flag-tag purified pilin is post-translationally modified. The LC-ESI/MS/MS analysis performed in this thesis using Flag-tag purified pilin successfully determined the phosphorylcholine post-translational modification sites. Based on the MS data and the mutagenesis analysis, phosphorylcholine is covalently linked to serine 157 and serine 160 of pilin. The colony immunoblot of a serine 157/160 to alanine mutant revealed that phosphorylcholine modifications of these sites on pilin are the only surface exposed phosphorylcholine and is responsible for binding to TEPC-15 (the monoclonal antibody which binds to phosphorylcholine). In this thesis, molecular modelling demonstrated that surface exposure of pilin phosphorylcholine could be altered by the phase variation of pilin glycosylation on the adjacent pilin monomer. Furthermore, the sites for phosphorylcholine modification are commonly observed in N. meningitidis strains but not in N. gonorrhoeae indicating the importance of phosphorylcholine in pathogenisis of N. meningitidis. In addition, the biosynthesis of phosphorylcholine for pilin post-translational modification still remains a mystery. Bacteria generally obtain choline from the environment. In this thesis, we demonstrated that pilin phosphorylcholine post-translational modification could be endogenously synthesized in N. meningitidis. In summary, this thesis describes the purification method of obtaining pure post-translationally modified pilin from N. meningitidis. The phosphorylcholine post-translation modification sites on pilin have been determined and showed the importance of these sites in antibody binding specificity.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 4 uppsatser.

Thesis (doctoral)--Lund University, 1997.

Thesis (doctoral)--Lund University, 1997.