Structural analysis of the master regulator Rns reveals a small molecule inhibitor of enterotoxigenic Escherichia coli virulence

Abstract

AbstractEnterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal disease worldwide and a frequent cause of travelers’ diarrhea. In addition to the production of enterotoxins, studies with human volunteers established ETEC virulence is dependent upon the production of proteinaceous adhesive pili for attaching to the intestinal wall. Although pilins are highly immunogenic, vaccines incorporating them have yet to be proven efficacious. An additional challenge for vaccines is the heterogeneity of ETEC pili, as 20 different pilus types have been identified. However, the expression of a significant number of pilus types is dependent upon Rns, an AraC family transcription factor. Furthermore, Rns also regulates the expression of the virulence factor CexE, an outer membrane coat protein. To determine how Rns functions and is regulated we solved its structure by X-ray crystallography to 3 Å resolution. Rns forms a dimer via its N-terminal domain and its structure is consistent with the dimer binding looped DNA. Our analyses also revealed a fatty acid, decanoic acid, bound within the Rns structure. Although Rns was not known to specifically bind small molecule ligands, biochemical analysis showed decanoic acid specifically stabilized Rns in a dose dependent manner. Lac reporter assays further showed that decanoic acid inhibits Rns function at both activated and repressed promoters. In situ, exogenous decanoic acid inhibited the expression of Rns-dependent CFA/I pili and CexE in different ETEC strains. Thus, our study reveals for the first time a naturally occurring small molecule ligand specifically inhibits Rns activity and potently suppresses the expression of ETEC virulence factors. Our findings provide an alternative approach to vaccines for inhibiting ETEC pathogenesis by using the Rns structure as a framework for rational drug design.