Identification of a novel zinc metalloprotease through a global analysis of Clostridium difficile extracellular proteins

PLoS One. 2013 Nov 26;8(11):e81306. doi: 10.1371/journal.pone.0081306. eCollection 2013.

Abstract

Clostridium difficile is a major cause of infectious diarrhea worldwide. Although the cell surface proteins are recognized to be important in clostridial pathogenesis, biological functions of only a few are known. Also, apart from the toxins, proteins exported by C. difficile into the extracellular milieu have been poorly studied. In order to identify novel extracellular factors of C. difficile, we analyzed bacterial culture supernatants prepared from clinical isolates, 630 and R20291, using liquid chromatography-tandem mass spectrometry. The majority of the proteins identified were non-canonical extracellular proteins. These could be largely classified into proteins associated to the cell wall (including CWPs and extracellular hydrolases), transporters and flagellar proteins. Seven unknown hypothetical proteins were also identified. One of these proteins, CD630_28300, shared sequence similarity with the anthrax lethal factor, a known zinc metallopeptidase. We demonstrated that CD630_28300 (named Zmp1) binds zinc and is able to cleave fibronectin and fibrinogen in vitro in a zinc-dependent manner. Using site-directed mutagenesis, we identified residues important in zinc binding and enzymatic activity. Furthermore, we demonstrated that Zmp1 destabilizes the fibronectin network produced by human fibroblasts. Thus, by analyzing the exoproteome of C. difficile, we identified a novel extracellular metalloprotease that may be important in key steps of clostridial pathogenesis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalysis
  • Clostridioides difficile / genetics
  • Clostridioides difficile / metabolism*
  • Enzyme Activation
  • Extracellular Space / metabolism
  • Fibrinogen / metabolism
  • Fibroblasts
  • Fibronectins / metabolism
  • Humans
  • Metalloproteases / chemistry
  • Metalloproteases / genetics
  • Metalloproteases / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Transport
  • Proteolysis
  • Proteomics*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Zinc / metabolism*

Substances

  • Bacterial Proteins
  • Fibronectins
  • Recombinant Proteins
  • Fibrinogen
  • Metalloproteases
  • Zinc

Grant support

The research was supported by Novartis Vaccines and Regione Toscana(BIOVAX project-POR CREO FESR 2007-20013) and MIUR (Italian Ministry of University and Research)[PON01_00117]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.