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 found in enterotoxigenic Escherichia coli (ETEC), a common cause of diarrheal disease. Similar proteins are present in other enteropathogens such as enteroaggregative E. coli and Citrobacter rodentium. Previous research revealed that the expression of CexE depends on Rns, a master virulence regulator belonging to the AraC/XylS superfamily of transcription factors.

In this investigation, the study found that genes cexD and cexPABC, responsible for lipidation and delivery of CexE to the outer membrane, are also regulated by Rns. Despite the genes being arranged in the same orientation, they are separated by a large intergenic region capable of accommodating a promoter and binding sites for transcription factors.

Nonetheless, the expression of the four-gene cluster does not initiate within this intergenic region. Instead, cexPABC genes are transcribed from the Rns-dependent cexE promoter, while cexD is expressed from its own Rns-dependent promoter. CexD is a unique bacterial acyltransferase that adds a lipid to the amino-terminal glycine of CexE, unlike typical acyltransferases that lipidate amino-terminal cysteines.

This lipidation process must occur in the periplasm, as topological mapping showed that CexD has six transmembrane domains with both its amino-terminus and enzymatic domain in the periplasm. Additionally, CexD is crucial for CexE localization to the outer leaflet of the outer membrane, and a cexD mutant of C. rodentium showed reduced pathogenicity in a murine model.

These findings expand the understanding of the Rns virulence regulon, the enzymology of a noncanonical acyltransferase, and its role in bacterial pathogenicity.