Identification and Characterization of Flagellar Co-expressed Determinants (Feds) of Campylobacter jejuni

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans throughout the world. In contrast to infection of humans, C. jejuni is a commensal organism of the intestinal tracts of wild and agriculturally-significant animals and avian species. Flagellar motility is the only virulence and colonization factor proven to be required for infection of human volunteers to promote disease and infection of poultry for commensalism. Expression of many flagellar genes is dependent on two alternative sigma factors, σ54 and σ28. Μany rod and hook genes are dependent on σ54 for expression, whereas σ28 is involved in the expression of the major flagellin and other filament genes. We investigated the σ28 regulon and identified five genes that are dependent on σ28 and flagellar components for maximal expression, but are not required for motility. One gene, ciaI, has previously been shown to function in intracellular survival after invasion of human intestinal epithelial cells. The four remaining genes, which we annotated as fedA-fedD (for flagellar co-expressed determinants), encode proteins that have not been characterized. Mutants lacking any one of these feds or ciaI demonstrated a reduced commensal colonization capacity in a natural chick model of colonization. Similar to the σ28-dependent gene product FspA1, a subset of these Feds is secreted by the bacterium in a flagellar-dependent manner. To further investigate the secretion requirements of these σ28-dependent proteins (FedB, CiaI and FspA1), we examined putative flagellar chaperones, flagellar components and other aspects of flagellar biosynthesis such as flagellar protein glycosylation for a role in secretion of Feds. We discovered that, like in other motile organisms, the FliJ chaperone is required for secretion of flagellar components in general, and that FliS is likely the chaperone for the major flagellin, FlaA. However, FliS and other putative flagellar chaperones are not required for secretion of the Feds. We also discovered that secretion of the Feds occurs during or just after hook biosynthesis, suggesting that construction of a hook is required for maximal secretion of these proteins via the flagellum. In addition, in the absence of the flagellar cap or flagellin glycosylation, we observed an increase in secretion of FedB, CiaI and FspA1, suggesting a possible inverse correlation between the amount of Fed proteins secreted via the flagella and length of the flagellar filament. Furthermore, we have identified N- and C-terminal intramolecular determinants within FedB and CiaI that are required for maximal secretion. Based on how other flagellar proteins are secreted, these findings indicate that a flagellar Type III secretion system (T3SS)-specific signal sequence is likely found at the N-terminus, and that an unidentified chaperone may bind to the C-terminus. Both of these factors appear to be required for maximal flagellar-dependent secretion of the Feds. We also examined the importance of secretion of Feds during commensal colonization and invasion of human colonic epithelial cells in vitro. Gentamicin-protection assays revealed that secretion of CiaI is not required for invasion of T84 cells. Furthermore, preliminary studies using a chick model of commensal colonization showed that secretion of FedB is important for colonization of the chick intestinal tract. However, whereas CiaI is required for colonization, secretion of CiaI was not important for colonization of the chick cecum. In summary, our work provides evidence that the flagellar system is a global regulatory system that coordinates production of flagella with colonization and virulence determinants, some of which are secreted in a flagellar-dependent manner, to promote maximal fitness during colonization and virulence.