Thèse de doctorat : Enzymologie Moléculaire : Nancy 1 : 2008. / Titre provenant de l'écran-titre.

Thèse de doctorat : Génie des procédés et des produits : INPL : 2008. / Titre provenant de l'écran-titre.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

Diarrheagenic Escherichia coli (E. coli) and Neisseria sp. are Gram-negative pathogens that cause high disease burden, especially in low-income countries.Enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) are a subset of E. coli that can cause disease. The sequence of E. coli genomes revealed the presence of at least 16 putative pili operons, it is still unknown if they encode functional pili. Several adhesins have been described in EPEC; however it is still an enigma if EHEC produces pili. In this dissertation the identification and characterization of a new pilus in EHEC is described. The main pilin subunit is encoded in the yagZ gene (renamed ecpA) and is present in all E. coli. We demonstrate ECP production in 137 (70%) of a total of 197 ecpA+ strains representing different categories of E. coli. Isogenic ecpA mutants of EHEC O157:H7 and fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Adherence levels were not hampered after single mutation of ecpA in EPEC. Only after the removal of the known EPEC adhesins such as BFP and intimin we were able to see significant reduction in adherence levels. In sum, ECP is the first pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization. However, EPEC-ECP plays a secondary role in adherence.Since 2007 the CDC recommends only third generation cephalosporins as the elected treatment for Neisseria gonorrhoeae infections. There is an urgent need to search for new drug targets and to development new drugs. Regions rich in guanine in the DNA are able to form secondary structures known as G-quadruplexes. It has been shown that G-quadruplexes are involved in control of transcription, translation and telomere elongation in mammalian cells. G-quadruplex interactive compounds are being developed for cancer therapy. G-quadruplex motifs are also present in bacteria. The fact that G-quadruplex interactive compounds can impair cancer development leads us to hypothesize that these drugs can be used as antimicrobials. This work presents evidence for the potential of G-quadruplex interactive compounds as broad-spectrum antimicrobials.

Neisseria gonorrhoeae infections are of major concern in areas of low socioeconomic status in both developed and developing nations. N. gonorrhoeae colonizes the genital tract by adhering to mucosal tissues through a number of adhesins, including the colony opacity-associated (Opa) proteins. Despite the random phase-variable expression of Opa proteins, 95% of clinical isolates express Opa variants that bind to the carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1), suggesting an essential role in vivo. Interestingly, even though gonorrhea is characterized by an intense inflammatory response, the organism is capable of evading the adaptive immune response. In previous studies by the Gray-Owen group, it has been established that certain gonococcal Opa variants bind CEACAM1 expressed by CD4+ T helper lymphocytes and, thereby, reduce their activation and proliferation. Since T cells are essential in establishing immune memory, inhibition of T cell function could explain the deficit in immune memory following gonococcal infection. In this thesis, I describe my studies to elucidate how CEACAM1 inhibits T cell activation on a molecular level. In Chapter 2, I demonstrate that outer membrane vesicles (OMVs) naturally shed by OpaCEA-expressing Neisseria sp. effectively inhibit CD4+ T cell activation, implicating a role for OMVs during infection and establishing that the Opa proteins do not have to be expressed in the context of the bacterium in order to elicit an inhibitory effect. In Chapter 3, I describe early steps in the CEACAM1-dependent inhibitory signaling cascade elicited when N. gonorrhoeae binds to CD4+ T cells. Finally, in Chapter 4, I show that a dynamic monomer-dimer equilibrium controls CEACAM1 function in epithelial cells and lymphocytes. Combined, the results presented in this thesis allow the derivation of a model to explain how CEACAM1 controls CD4+ T cell function at a molecular level.

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.

This thesis describes the construction and evaluation of six knockout mutants in Neisseria meningitidis serogroup B strain MC58. The genes that were inactivated were NMB0711, NMB1048, NMB1525, NMB1898, NMB1948 and NMB1966. Attempts to inactivate a seventh gene, NMB0768, were not successful. These genes were chosen as they had been observed previously to be up-regulated following incubation in whole blood using Differential Fluorescence Induction. Mutant strains were constructed by allelic exchange with a plasmid construction in which a kanamycin resistance cassette had been incorporated within the coding sequence of each cloned target gene. Confirmation of successful allelic exchange was achieved by Southern blotting. The phenotype of all mutant strains were evaluated by assessment of in vitro growth and in the infant mouse model of infection. Of the six mutants, all except that involving NMB1966, showed no differences compared with wild-type. The mutant knockout of NMB1966 showed (1) impaired growth beyond the mid-logarithmic phase in shaking broth culture but normal growth on solid medium, (2) reduced virulence in a mouse model of infection, (3) impairment in its capacity to invade (although not adhere to) cultured human bronchial epithelial cells, and (4) more rapid killing in ex vivo human blood. NMB1966 is predicted to encode the ATP-binding subunit of an ABC transporter and, after experiments for this thesis had been completed, it was demonstrated, by others, that this ABC transporter is responsible for uptake of L-Glutamate at low sodium concentrations. It is likely that defective uptake of L-Glutamate explains the observed defect in shaking broth culture and intracellular survival, both of which are associated with low ambient concentrations of sodium. However, it is not certain if this mechanism explains the observed defect in survival in human blood and in the infant mouse model which test predominantly extracellular survival and represent environments with high sodium concentrations.

Würzburg, Universiẗat, Diss., 2008.

Würzburg, Univ., Diss., 2005. / Erscheinungsjahr an der Haupttitelstelle: 2004