Regulation and Characterization of the Cex system of enteric pathogens: a pathway for the lipidation and secretion of noncanoncial bacterial lipoproteins.

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.