Etude fonctionnelle de l'operon fimbriaire stg de Salmonella enterica serovar Typhi

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans and is considered as an important health problem. Most of our knowledge on the pathogenesis of this bacterium comes from an enteric fever model in mice caused by serovar Typhimurium. Few studies have examined the virulence factors unique to serovar Typhi or the possibility that pseudogenes harbored in its genome may be functional. stg fimbriae are found only within the serovar Typhi genome and contain a premature TAA stop codon in the stgC gene encoding the usher responsible for the assembly of fimbrial subunits at the bacterial surface. Thus, the stg fimbria has been classified among the list of non-functional pseudogenes. The objectives of this study were to assess the involvement of stg fimbriae during interaction with human cells, and then to evaluate the importance of the stgC pseudogene in fimbrial biogenesis.;First, stg transcription was evaluated using a lacZ fusion. Despite low expression levels generally observed in rich medium, growth in minimal medium promoted transcription of the operon. Complete deletion of the stgABCD fimbrial operon from S. Typhi was performed by allelic exchange and was complemented on a plasmid. It has been shown that the presence of stg in S. Typhi, S. Typhimurium and E. coli contributes to increased adherence to human epithelial cells. In addition, the fimbriae seem to act as an anti-phagocytic structure during the interaction with macrophages. Thus, the stg operon appears to be functional despite its premature codon, as phenotypes were observed.;The second part of this study involved testing the role of the stgC pseudogene in fimbrial biogenesis. Different variants of the operon were generated, cloned into an arabinose inducible vector, and then transformed into afimbriated E. colii strain ORN172. Translocation of the StgD subunit to the cell surface of the different mutants was evaluated using Western blot. This experiment demonstrated that stgC is essential for export of the StgD subunit to the cell surface. The addition of a 6-histidine tag at the C-terminal end of StgC confirmed the complete translation of the gene, despite the premature TAA stop codon. Peptide sequencing revealed the insertion of a tyrosine at this codon. A translational fusion with the green fluorescent protein demonstrated that approximately 0.8% of the mRNA can be translated to allow full production of the usher.;This project allowed characterization of a virulence factor unique to S. Typhi and is a step closer towards better understanding of its pathogenesis mechanisms. This is the first demonstration in bacteria of the functionality of a gene which is interrupted by a premature TAA stop codon.;Keywords : Salmonella enterica serovar Typhi, fimbriae, Stg, pathogenesis, pseudogene...