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: The ZmpI cleavage sites within CD2831 are not randomly distributed along the protein sequence but are confined in the C-terminal extremity, directly upstream of the putative PPKTG sorting motif (30). Thus, it is possible that the CD2831 protein is anchored to the cell wall through a sorting reaction catalyzed by SrtB and subsequently secreted into the extracellular medium after its cleavage by the ZmpI protease. To test this hypothesis, the srtB gene in the ΔzmpI mutant strain was inactivated by deletion. pJKP041 was then introduced into the ΔzmpIΔsrtB double mutant, and the subcellular localization of CD2831 in this strain grown in the presence of inducer was then analyzed by immunoblotting and immunofluorescence microscopy. Cell wall extracts from the ΔzmpIΔsrtB mutant displayed undetectable levels of CD2831, whereas the protein was strongly detected in the supernatant fraction (Fig. 4). In agreement with these data, the anti-CD2831 antibodies did not surface-label the ΔzmpIΔsrtB mutant strain (Fig. 3), demonstrating that SrtB is required for anchoring of CD2831 to the cell wall.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.