Regulation and Characterization of the Cex system of enteric pathogens: a pathway for the lipidation and secretion of noncanoncial bacterial lipoproteins
TL;DR
It is shown that CexD is required for CexE’s localization to the outer leaflet of the outer membrane and that a cexD mutant of C. rodentium is attenuated in a murine model, which further define the Rns virulence regulon, the enzymology of a noncanonical acyltranferase and its contributions to pathogenicity.
Abstract
CexE is an outer membrane lipoprotein of enterotoxigenic Escherichia coli (ETEC); one of the most prevalent etiological agents of diarrheal disease. Homologs of CexE are present in enteroaggregative E. coli, Citrobacter rodentium, and other enteropathogens. We have previously shown that expression of CexE is dependent upon Rns, the master virulence regulator of ETEC and a member of the AraC/XylS superfamily of transcription factors. In this study we report that the expression of cexD and cexPABC, the genes required for lipidation and delivery of CexE to the outer leaflet of the outer membrane, are also Rns-dependent. Although cexE and cexPABC are arranged in the same orientation, they are separated by an intergenic region sufficiently large to accommodate a promoter and transcription factor binding sites. Nevertheless, expression of the four gene cluster does not originate within the intergenic region. Rather, cexPABC are expressed from the Rns-dependent cexE promoter. The remaining gene of the system, cexD, is monocistronic and expressed from its own Rns-dependent promoter. Mutagenesis of a predicted Rns binding site upstream of cexD abolished Rns binding to a cexD promoter fragment in vitro and activation of the promoter in vivo. CexD is a novel bacterial acyltransferase. Unlike canonical bacterial acyltransferases that lipidate amino-terminal cysteines, CexD attaches a lipid to the amino-terminal glycine of CexE. Lipidation must occur in the periplasm because topological mapping revealed that CexD has six transmembrane domains with both its amino-terminus and enzymatic domain in the periplasm. We further show that CexD is required for CexE’s localization to the outer leaflet of the outer membrane and that a cexD mutant of C. rodentium is attenuated in a murine model. These findings further define the Rns virulence regulon, the enzymology of a noncanonical acyltranferase and its contributions to pathogenicity.