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 cell wall fraction of C. difficile expressing CD2831R-HA was solubilized by digestion with CD27L, a C. difficile phage-endolysin predicted to have N-acetylmuramoyl-l-amidase activity (Fig. 5B) (22, 33). Cell wall-anchored CD2831R-HA was then purified from this digest by immunoprecipitation with an anti-HA affinity resin (Fig. 5C). The enriched protein was subjected to digestion with the endopeptidase Arg-C, which cleaves at the C terminus of arginine residues, and C-terminal peptides harboring the HA tag were purified again by immunoprecipitation. The structures of the resulting eluted peptides, expected to be covalently anchored to peptidoglycan peptide side chains, were then analyzed by a combination of LC-MS and MS/MS. All peptides corresponding to CD2831 eluted as a single wide peak (Fig. 6A). The seven prominent ions contained in this peak were assigned based on their mass determination to structures in agreement with an HA-tagged C-terminal CD2831 peptide that terminates at the threonine residue of the PPKTG sorting motif and is covalently linked to the peptide side chain of peptidoglycan (Fig. 6, B and C). Among the different ion signals observed, the predominant ion at m/z 802.06 with z = 3 and the minor ion at m/z 1202.59 with z = 2 are proposed to have identical structures, consisting of the anchor peptide linked to cell wall tripeptide (d-Glu-(YPYDVPDYAVNPPVPPKT-)mDAP-d-Ala). Both ions were subjected to MS/MS analysis, and the main daughter ions were assigned to the corresponding peptide structure (Figs. 6D and 7A and Tables 1 and 2). In both cases, the fragmentation pattern was consistent with the predicted structure and thus confirmed that CD2831 is covalently anchored to the peptidoglycan. The alanine residue in the cell wall tripeptide was unambiguously found to be located at the C terminus of the peptide chain of the peptidoglycan rather than at its N terminus. Indeed, fragmentation of the parental ion at m/z 802.06 (z = 3) led to the generation of several daughter ions lacking the glutamate residue but not the alanine residue (fragments with an m/z 489.86, z = 2; m/z 685.26, z = 1; m/z 759.11, z = 1; m/z 881.40, z = 1; and m/z 978.39, z = 1) and to the generation of a daughter ion at m/z 772.43 (z = 3) corresponding to the loss of an alanine residue (−89.05 Da) from the C-terminal end of the peptide stem. Moreover, because either the glutamate or alanine residue could be individually lost, this result strongly suggests that the anchor peptide is linked to the mDAP residue of the peptide side chain of peptidoglycan. The minor ion at m/z 808.05 (z = 3) had a 17.95- Da mass difference compared with the predominant ion signal, suggesting the addition of a water molecule (Fig. 6, B and C). MS/MS fragmentation of this ion revealed that this additional mass could be easily lost as most of the daughter ions presented two different forms, with or without the extra 18 Da. Moreover, this mass could be lost either from the N-terminal extremity of the anchor peptide or from the peptidoglycan peptide stem (Fig. 7B and Table 3). Therefore, the ion at m/z 808.05 (z = 3) corresponds to the H+ + H2O adduct of the main structure identified above.
Affiliation: From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.