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

摘要

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 IIAGlc (EIIAGlc) interacts with a peptidase that has high sequence similarity to mammalian insulin-degrading enzymes, called Vibrio insulin-degrading enzyme (vIDE). Although the vIDE–EIIAGlc interaction is independent of the phosphorylation state of EIIAGlc, vIDE shows no peptidase activity unless complexed with the unphosphorylated form of EIIAGlc. 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: PL for the longer transcript and PS 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 PS transcription in the presence of cAMP whose concentration is regulated by EIIAGlc. These results suggest that EIIAGlc regulates the expression level of vIDE as well as its activity.