Investigation of factors required for assembly of the GspD secretin in Escherichia coli and Vibrio species

The type II secretion system (T2SS) is a major virulence factor in Gram-negative bacteria due to the involvement of this system in the pathogenesis of numerous species. This specialized macromolecular apparatus spans the cell envelope and functions to translocate folded proteins located in the periplasm across the outer membrane. In the pathogens enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae, the T2SS is integral for infection of the host because it serves as the conduit for secretion of the heat-labile enterotoxin (LT) and the cholera toxin respectively. The objective of this study was to assess the assembly and function of the T2SSs in ETEC and Vibrio cholerae by genetic, physiological and molecular biological techniques. In silico analysis of the ETEC H10407 genome identified two T2SSs designated alpha and beta. The T2SSα is not assembled in ETEC under standard laboratory conditions. Replacement of the cryptic endogenous gspAα and gspCα promoters with inducible ones resulted in assembly of the T2SSα, however the system remained incapable of secreting LT. Under laboratory conditions the GspDβ secretin of the T2SSβ was readily detectable and expression of a functional T2SSβ was required for secretion of LT into culture supernatant. The requirement for T2SS accessory proteins GspA, GspB, GspS and LeoA in GspDβ secretin assembly was investigated. The hypothetical lipoprotein YghG (renamed GspSβ) encoded in the T2SSβ operon of ETEC was characterized as a pilotin protein required for localization of the secretin protein GspDβ to the outer membrane, since in the absence of the lipoprotein GspDβ was localized to the inner membrane in monomeric form and mostly degraded. The hypothetical virulence factor LeoA was shown to be not required for GspDβ secretin assembly or LT secretion. In Vibrio cholerae, the peptidoglycan-remodeling complex GspAB was shown to be partially required for GspDVc secretin assembly, suggesting the possibility that another protein, possibly the ETEC YghG homologue, is also required for GspDVc secretin assembly in Vibrio species. Lastly, a novel application of the bacterial two hybrid technique was developed that enabled screening for interactions with a protein of interest with proteins encoded by the entire coding capacity of the ETEC H10407 genome. In particular, a library composed of 6.2 x 105 plasmids comprised of overlapping ETEC genomic fragments was encoded in the bacterial two-hybrid pTRG vector and was screened for interactions with members of the ETEC T2SS