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.

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.

Fímbrias do tipo IV estão associadas a diversos fenótipos em bactérias gram-negativas, e o presente estudo consistiu na caracterização da fímbria Pil e investigação de seu papel na adesão bacteriana de isolados de EPEC atípica. Por PCR e RT-PCR foram investigadas a presença e a funcionalidade do operon Pil e os resultados demonstraram que este está sendo transcrito somente nos isolados BA558 e BA956. Os genes pilS e pilV foram clonados em vetor de expressão para obtenção das proteínas Pil recombinantes e produção de anticorpos policlonais. A análise qualitativa dos testes de inibição da adesão utilizando os soros anti-PilS e anti-PilV juntos demonstraram que o isolado BA558 apresentou mudança de fenótipo de adesão. Esses resultados nos permitem concluir que o operon Pil está funcional em BA558 e BA956, e a expressão da fímbria Pil nessas cepas não está relacionada à formação de biofilme e autoagregação, porém a proteína fimbrial PilS juntamente com a adesina PilV parecem exercer uma função acessória importante na interação de BA558 às células HEp-2. / Type IV fimbriae are associated with several phenotypes in gram-negative bacteria. The aim of this study was the characterization of the Pil fimbria and its role in the interaction of atypical EPEC isolates in bacterial adhesion. Using PCR and RT-PCR, we investigated the presence and functionality of the pil operon genes. The results showed that these genes are transcribed only in the BA558 and BA956 isolates. The pilS and pilV genes were cloned into an expression vector for recombinant proteins and polyclonal antibodies production. Qualitative analysis of the adherence inhibition assays using both rabbit sera changed to localized-like the phenotype of BA558 isolate adhesion. Together, these results allow us to conclude that the Pil operon is functional only in the BA558 and BA956 isolates and that the expression of Pil fimbriae in aEPEC is not related to biofilm formation and autoaggregation but, the fimbrial PilS protein together with PilV adhesin seem to play an important accessory function in the interaction between the BA558 and epithelial cells in vitro.