Cyclic diGMP Regulates Production of Sortase Substrates of Clostridium difficile and Their Surface Exposure through ZmpI Protease-mediated Cleavage

• Published in: The Journal of biological chemistry Vol. 290; no. 40; pp. 24453 - 24469
• Main Authors: Peltier, Johann, Shaw, Helen A., Couchman, Edward C., Dawson, Lisa F., Yu, Lu, Choudhary, Jyoti S., Kaever, Volkhard, Wren, Brendan W., Fairweather, Neil F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-10-2015; American Society for Biochemistry and Molecular Biology
• Subjects: Adhesins, Bacterial - metabolism; Amino Acid Motifs; Aminoacyltransferases - metabolism; bacterial pathogenesis; Bacterial Proteins - metabolism; Cell Membrane - metabolism; cell surface protein; Cell Wall - metabolism; cell wall anchoring; Clostridioides difficile - metabolism; Clostridium difficile; cyclic diGMP (c-diGMP); Cyclic GMP - analogs & derivatives; Cyclic GMP - chemistry; Cysteine Endopeptidases - metabolism; Gene Expression Profiling; Gene Expression Regulation, Bacterial; gene regulation; Metalloproteases - metabolism; Microbiology; Microscopy, Fluorescence; Mutation; Oligonucleotides - metabolism; Peptide Hydrolases - metabolism; Peptidoglycan - chemistry; Plasmids - metabolism; Protein Binding; Protein Structure, Tertiary; proteinase; sortase; Tandem Mass Spectrometry; Virulence Factors - metabolism; cell surface protein; proteinase; cyclic diGMP (c-diGMP); gene regulation; sortase; Clostridium difficile; cell wall anchoring; bacterial pathogenesis
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.665091

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro, and seven potential substrates have been identified. Here, we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. 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. We show that CD2831 protein levels are elevated in the presence of high intracellular cyclic diGMP (c-diGMP) concentrations, in agreement with the control of CD2831 expression by a c-diGMP-dependent type II riboswitch. Low c-diGMP levels induce the release of CD2831 and presumably CD3246 from the surface of cells. 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.
Background: Bacteria use various mechanisms to anchor their surface proteins, including a sortase enzyme.
Results: Covalent anchoring of proteins to the peptidoglycan in Clostridium difficile and its regulation by cyclic diGMP and protease activity are demonstrated.
Conclusion: A novel regulatory mechanism of cell wall protein anchoring is found.
Significance: Elucidating how proteins are anchored may shed light on control of bacterial colonization in vivo.