Clostridium difficile secreted Pro‐Pro endopeptidase PPEP‐1 (ZMP1/CD2830) modulates adhesion through cleavage of the collagen binding protein CD2831

Abstract

a b s t r a c tThe Clostridium difficile cd2830 gene product is a secreted metalloprotease, named Pro-Pro endopeptidase (PPEP-1). PPEP-1 cleaves C. difficile cell surface proteins (e.g. CD2831). Here, we confirmed that PPEP-1 has a unique preference for prolines surrounding the scissile bond. Moreover, we show that it exhibits a high preference for an asparagine at the P2 position and hydrophobic residues at the P3 position. Using a PPEP-1 knockout C. difficile strain, we demonstrate that the removal of the collagen binding protein CD2831 is fully attributable to PPEP-1 activity. The PPEP-1 knockout strain demonstrated higher affinity for collagen type I with attenuated virulence in hamsters.

Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection

“…A number of C. difficile virulence factors have a Zn requirement; however, the impact of Zn on CDI has yet to be explored 14–17 . To test this, we used a mouse model of CDI that induces susceptibility to C. difficile through administration of cefoperazone (0.5 mg/ml)(Supplementary Fig.…”

Covalent attachment and Pro‐Pro endopeptidase (PPEP‐1)‐mediated release of Clostridium difficile cell surface proteins involved in adhesion

“…) homologous to the Staphylococcus aureus CNA collagen binding domain (PDB ID: 2F6A). Indeed, binding of CD2831 to collagen has been demonstrated in vitro (Hensbergen et al ., ). CD3392 and CD0386 contain putative Cna B domains, which are found in collagen binding proteins but are not directly involved in the binding but more in the projection of the actual collagen binding domain.…”

“…Hence, the name Pro‐Pro endopeptidase, PPEP‐1 (EC 3.4.24.89; formerly Zmp 1 and CD2830) was coined for this enzyme (Fig. ) (Hensbergen et al ., ; Hensbergen et al ., ).…”

“…Indeed, endogenous PPEP‐1 mediated cleavage of CD2831 and CD3246 could be demonstrated using mass spectrometric analysis of product peptides within conditioned medium of C. difficile cells. Importantly, this cleavage was only observed in WT cells and was absent in ppep‐1 knockout cells, demonstrating the direct link between PPEP‐1 activity and cleavage of these proteins (Hensbergen et al ., ). Of note, the PPEP‐1 activity is sufficient to completely remove CD2831 from the cell surface when cells are in the stationary phase during in vitro growth (Hensbergen et al ., ), even in a system where CD2831 is overexpressed (Peltier et al ., ).…”

“…Importantly, this cleavage was only observed in WT cells and was absent in ppep‐1 knockout cells, demonstrating the direct link between PPEP‐1 activity and cleavage of these proteins (Hensbergen et al ., ). Of note, the PPEP‐1 activity is sufficient to completely remove CD2831 from the cell surface when cells are in the stationary phase during in vitro growth (Hensbergen et al ., ), even in a system where CD2831 is overexpressed (Peltier et al ., ). Moreover, uncleaved endogenous CD2831 in the ppep‐1 knockout could be removed from the bacterial surface by shaving it off with recombinant PPEP‐1, further confirming the vital role for PPEP‐1 in this cleavage (Hensbergen et al ., ).…”