Cyclic diGMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage.
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.
From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
Johann
J
Peltier
From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
Helen A
HA
Shaw
From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom.
Edward C
EC
Couchman
the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.
Lisa F
LF
Dawson
the Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom, and.
Lu
L
Yu
the Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom, and.
Jyoti S
JS
Choudhary
the Research Core Unit Metabolomics, Hannover Medical School, Hannover D-30625, Germany.
Volkhard
V
Kaever
the Department of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom.
Brendan W
BW
Wren
From the Department of Life Sciences, Center for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom, n.fairweather@imperial.ac.uk.
Neil F
http://orcid.org/0000-0002-3224-0207
NF
Fairweather
D018829:Adhesins, Bacterial / Q000378:metabolism; D020816:Amino Acid Motifs; D019881:Aminoacyltransferases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism*; D016360:Clostridioides difficile / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000378:metabolism; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D045726:Metalloproteases / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053719:Tandem Mass Spectrometry; D037521:Virulence Factors / Q000378:metabolism
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D009841:Oligonucleotides; D010457:Peptidoglycan; D037521:Virulence Factors; C062025:bis(3',5')-cyclic diguanylic acid; D019881:Aminoacyltransferases; D045726:Metalloproteases; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D006152:Cyclic GMP
Clostridium difficile; bacterial pathogenesis; cell surface protein; cell wall anchoring; cyclic diGMP (c-diGMP); gene regulation; proteinase; sortase