A Novel Secreted Metalloprotease (CD2830) from Clostridium difficile Cleaves Specific Proline Sequences in LPXTG Cell Surface Proteins

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 HSP90␤, 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 Clostridium difficile is an anaerobic, Gram-positive, sporeforming bacterium. Human intake of spores occurs through the fecal-oral route. Upon leaving the stomach and becoming exposed to bile acids in the small intestine, the C. difficile spores germinate. Once germinated, the vegetative cells in the colon encounter an unreceptive environment (1). Competition with the normal flora, immune responses, gastric fluids (2), and specialized antimicrobial peptides all act against a developing infection. Moreover, the physical barrier formed by a layer of glycoproteins (mucins) covering the underlying epithelial cells forms a major hurdle for firm adhesion.Many enteric pathogens express factors that reduce competition, allow evasion of host immune responses, and promote adhesion and/or invasion of tissues. These virulence factors are located in the cell membrane or wall (controlling adhesion and protection) or are secreted (modifying the surrounding environment). The best studied virulence factors in C. difficile are the toxins TcdA and TcdB (3, 4), which cause destruction of the intestinal barrier by disrupting the epithelial actin cytoskeleton. It is speculated that the subsequent increased permeability of the intestinal epithelium leads to increased exudation of fluids, including nutritional substances. Damage to the intestinal mucosa causes the main symptoms of C. difficile infection, including pseudomembranous colitis (1).Data illustrating how C. difficile circumvents host defense mechanisms are limited. As an example, in response to attack by antimicrobial peptides, C. difficile expresses a set of genes that change the surface charge, thereby diminishing the interaction of cationic antimicrobial peptides on th...

Cyclic Di-GMP Riboswitch-Regulated Type IV Pili Contribute to Aggregation of Clostridium difficile

“…The antagonistic regulation by c-di-GMP of motility and sessile behaviors, namely, aggregation and biofilm formation, was also recently reported by Souturina et al (32). The zinc metalloprotease Zmp1 encoded by CD2830, which is located downstream of the c-di-GMP-I riboswitch Cdi1_12, was recently found to cleave fibronectin, fibrinogen (33), the putative adhesin CD2831, and the putative surface protein CD3246, the last two encoded by genes located downstream of the c-di-GMP-II riboswitches Cdi2_3 and Cdi2_1, respectively (34).…”

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

“…The plasmid was introduced by conjugation into the CT::PPEP-1 knockout strain. The PPEP-1 expression construct was made, and PPEP-1 was purified, as described previously [14].…”

“…Intriguingly, two adjacent genes cd2830 and cd2831, are inversely regulated by c-di-GMP [12]. cd2830 encodes a secreted zinc metalloprotease [14,15] and cd2831 a putative collagen binding protein. two prolines [14].…”

“…We have also identified CD2831 and CD3246 as genuine substrates of PPEP-1. Remarkably CD2831 and CD3246 contain six and seven consecutive cleavage sites, respectively [14], located directly adjacent to the (S/P)PXTG peptidoglycan cell wall anchor motif of these surface proteins [14,19].…”

“…cd2830 encodes a secreted zinc metalloprotease [14,15] and cd2831 a putative collagen binding protein. two prolines [14]. Efficient proteolytic cleavage between two prolines that are not located close to the protein N-terminus have long been thought to be almost impossible [16,17] and CD2830 is the first enzyme that is designed for this purpose.…”