Related Experiment Video
Updated: May 2, 2026
Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Published on: December 30, 2016
A novel secreted metalloprotease (CD2830) from Clostridium difficile cleaves specific proline sequences in LPXTG cell
Paul J Hensbergen1, Oleg I Klychnikov, Dennis Bakker
1Center for Proteomics and Metabolomics, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands;
Abstract:
Bacterial secreted proteins constitute a biologically important subset of proteins involved in key processes related to infection such as adhesion, colonization, and dissemination. Bacterial extracellular proteases, in particular, have attracted considerable attention, as they have been shown to be indispensable for bacterial virulence. Here, we analyzed the extracellular subproteome of Clostridium difficile and identified a hypothetical protein, CD2830, as a novel secreted metalloprotease. Following the identification of a CD2830 cleavage site in human HSP90β, a series of synthetic peptide substrates was used to identify the favorable CD2830 cleavage motif. This motif was characterized by a high prevalence of proline residues. Intriguingly, CD2830 has a preference for cleaving Pro-Pro bonds, unique among all hitherto described proteases. Strikingly, within the C. difficile proteome two putative adhesion molecules, CD2831 and CD3246, were identified that contain multiple CD2830 cleavage sites (13 in total). We subsequently found that CD2830 efficiently cleaves CD2831 between two prolines at all predicted cleavage sites. Moreover, native CD2830, secreted by live cells, cleaves endogenous CD2831 and CD3246. These findings highlight CD2830 as a highly specific endoproteinase with a preference for proline residues surrounding the scissile bond. Moreover, the efficient cleavage of two putative surface adhesion proteins points to a possible role of CD2830 in the regulation of C. difficile adhesion.
Insights
Clostridium difficile secretes a novel metalloprotease, CD2830, that uniquely cleaves Pro-Pro bonds. This protease targets adhesion molecules, suggesting a role in regulating bacterial adherence during infection.
Area of Science:
- Microbiology
- Proteomics
- Molecular Biology
Background:
- Bacterial secreted proteins are crucial for infection processes like adhesion and dissemination.
- Extracellular proteases play a significant role in bacterial virulence.
Purpose of the Study:
- To identify and characterize novel secreted proteins from Clostridium difficile.
- To investigate the function and substrate specificity of the identified metalloprotease CD2830.
Main Methods:
- Analysis of the Clostridium difficile extracellular subproteome.
- Identification of a hypothetical protein, CD2830, as a secreted metalloprotease.
- Determination of CD2830 cleavage motif using synthetic peptide substrates.
- In vitro and in vivo cleavage assays of putative adhesion molecules (CD2831, CD3246).
Main Results:
- CD2830 was identified as a novel secreted metalloprotease.
- CD2830 exhibits a unique preference for cleaving Pro-Pro bonds.
- Two Clostridium difficile adhesion molecules, CD2831 and CD3246, contain multiple CD2830 cleavage sites.
- CD2830 efficiently cleaves these adhesion molecules, both in vitro and in vivo.
Conclusions:
- CD2830 is a highly specific endoproteinase with a unique proline-directed cleavage activity.
- The cleavage of adhesion molecules by CD2830 suggests a role in regulating bacterial adhesion and potentially virulence.
- This discovery provides new insights into the molecular mechanisms of Clostridium difficile pathogenesis.
