Flagellins are proteins of microbial origin that confer motility to bacteria. They exhibit a stupendous conservation in their structure, required for assembly into polymeric flagella. The various environmental and evolutionary pressures encountered by motile bacteria have however generated a multiplicity of primary protein sequences. In mammals, specific receptors, namely TLR5 and Ipaf, are devoted to the immunosurveillance of flagellins. We sought to explore the diversity of flagellin structures expressed by the intestinal microbiota and how these proteins may influence the host innate immune response. In this context, recombinant flagellins derived from Gram-negative and Gram-positive, pathogenic and commensal bacteria from the γ-Proteobacteria and the Clostridium sub-phyla were expressed in E.coli and then purified. Protein sequence alignment and phylogenetic analysis of twelve flagellins revealed clustering dependent on three criteria: the phylum, the genus and the commensal or pathogenic nature of the bacteria. We next characterized these structures functionally using in vitro cell systems including epithelial and dendritic cells. In a TLR5-dependent manner, flagellin induced the secretion of pro-inflammatory chemokines in intestinal epithelial cells, which are considered crucial in triggering the recruitment of immune cell effectors. Of importance, the secretion levels were directly dependent of the flagellin structure, as confirmed by the various agonistic potentials determined in dose-response experiments. We observed that commensal flagellins were in general less potent than pathogenic flagellins. Similarly, the effects on dendritic cells following flagellin exposure were ligand-dependent. Overall, TLR5 activation constitutes a ligand-dependent TLR5-flagellin complex formation with sequential activation of MAPK and/or NF-kB and signalling, resulting in differential modulation of the intracellular signalling cascade downstream. The results presented in this thesis suggest innate signalling mediated by flagellated pathogens and commensals may affect the intestinal immune status in a number of distinct ways, either by promoting active immune response or by promoting tolerance response.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:582682 |
| Date | January 2013 |
| Creators | Monnais, Edouard |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=202130 |
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