Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.
Bottom Line: The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues, and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem.This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type I c-diGMP riboswitch.These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-diGMP, on which surface anchoring is dependent.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.Show MeSH
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Mentions: Seven putative sortase substrates have been identified in C. difficile 630, but only four are conserved in the ribotype 027 strain R20291 (17). Their putative functions are as follows: CD0183 and CD2768, cell wall hydrolases; CD2537, a 5′-nucleotidase/phosphoesterase; and CD2831, a collagen-binding protein. CD2831 contains two collagen-binding domains and a CnaB-like domain (Fig. 2A). CnaB domains, first identified in S. aureus (28), are commonly found in cell-surface adhesins and are thought to form a stalk presenting the ligand-binding domain away from the bacterial cell surface (29). CD2831 is efficiently cleaved in vitro by the secreted zinc metalloprotease ZmpI (CD2830) (30, 31). Six different ZmpI target cleavage sites have been identified within the C terminus of CD2831 (Fig. 2A) and a proteomic analysis of the extracellular medium of C. difficile cultures revealed that CD2831 is at least partly secreted (30). To give further insight into the cellular localization of CD2831, immunoblotting was used to localize the protein in cellular fractions and culture supernatants of several strains. CD2831 was poorly detected in both the supernatant and in whole cell lysate of strain 630 (Fig. 2B and data not shown), suggesting that the normal levels of CD2831 are low. Therefore, the CD2831 gene was cloned under control of the inducible Ptet promoter on a plasmid, yielding pJKP041. Strain 630 (pJKP041) was grown in the presence of inducer, and the distribution of CD2831 in supernatant, cell wall, membrane, and cytoplasmic fractions was investigated. Two polypeptides were detected by Western blotting, observed mainly in the culture supernatant (Fig. 2B). Because SrtB expression was undetectable in the wild-type 630 strain, secretion of the putative sortase substrate CD2831 into the culture supernatant could be due to the absence of sortase, leading to the inability to anchor CD2831 to the cell surface. Therefore, pJKP041 was introduced into the Ptet-srtB strain, and the resultant strain was grown in the presence of inducer. However, the subcellular distribution of CD2831 remained unchanged in this strain (Fig. 2B), demonstrating that the secretion of CD2831 into the culture supernatant is independent of SrtB.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.