Expression of Vibrio vulnificus insulin-degrading enzyme is regulated by the cAMP–CRP complex

Abstract

Components of the bacterial phosphoenolpyruvate (PEP) : carbohydrate phosphortransferase system (PTS) have multiple regulatory roles in addition to PEP-dependent transport/phosphorylation of numerous carbohydrates. We have recently shown that, in an opportunistic human pathogen, Vibrio vulnificus, enzyme IIA(Glc) (EIIA(Glc)) interacts with a peptidase that has high sequence similarity to mammalian insulin-degrading enzymes, called Vibrio insulin-degrading enzyme (vIDE). Although the vIDE-EIIA(Glc) interaction is independent of the phosphorylation state of EIIA(Glc), vIDE shows no peptidase activity unless complexed with the unphosphorylated form of EIIA(Glc). A deletion mutant of ideV, the gene encoding vIDE, shows remarkably lower degrees of survival and virulence than the wild-type strain in mice, implying that vIDE is a virulence factor. In this study, we investigated regulation of ideV expression at the transcriptional level. Primer extension analysis identified two different transcriptional start sites of ideV: P(L) for the longer transcript and P(S) for the shorter transcript. We performed ligand fishing experiments by using the promoter region of ideV and found that the cAMP receptor protein (CRP) specifically binds to the promoter. DNase I footprinting experiments revealed that CRP binds to a region between the two promoters. In vitro transcription assays showed that CRP activates ideV P(S) transcription in the presence of cAMP whose concentration is regulated by EIIA(Glc). These results suggest that EIIA(Glc) regulates the expression level of vIDE as well as its activity.