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: To assess CD2831 localization at the individual cell level, immunofluorescence microscopy analysis was performed on intact bacteria. All bacteria were uniformly labeled with anti-LMW SLP, which detects the S-layer that completely coats C. difficile (Fig. 3). CD2831 could be detected at the surface of the induced ΔzmpI pJKP041 strain (Fig. 3), but the intensity of labeling by the anti-CD2831 antibodies appeared to be heterogeneous in the bacterial population. Interestingly, CD2831 labeling was not distributed along the entire bacterial cell but was concentrated mostly at one cell pole. Similar features have been previously reported for SrtB substrates in Listeria monocytogenes (see below) (32). Most cells from the induced wild-type pJKP041 culture were not stained with anti-CD2831, with the exception of polar staining in a few cells. This residual fluorescence was not observed in the uninduced ΔzmpI pJKP041 strain, indicating that a small fraction of CD2831 remains at the cell surface in the induced wild-type pJKP041 strain. It is likely that the level of ZmpI in the extracellular medium is insufficient to cleave efficiently the large amount of CD2831 protein produced under inducing conditions.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.