Purification and characterization of a clostripain-like protease from a recombinant Clostridium perfringens culture

Abstract

Clostridium perfringens produces a homologue of clostripain (Clo), the arginine-specific endopeptidase of Clostridium histolyticum. To determine the biochemical and biological properties of the C. perfringens homologue (Clp), it was purified from the culture supernatant of a recombinant C. perfringens strain by cation-exchange chromatography and ultrafiltration. Analysis by SDS-PAGE, N-terminal amino acid sequencing and TOF mass spectrometry revealed that Clp consists of two polypeptides comprising heavy (38 kDa) and light (16 kDa or 15 kDa) chains, and that the two light chains differ in the N-terminal cleavage site. This difference in the light chain did not affect the enzymic activity toward N-benzoyl-L-arginine p-nitroanilide (Bz-L-arginine pNA), as demonstrated by assaying culture supernatants differing in the relative ratio of the two light chains. Although the purified Clp preferentially degraded Bz-DL-arginine pNA rather than Bz-DL-lysine pNA, it degraded the latter more efficiently than did Clo. Clp showed 2.3-fold higher caseinolytic activity than Clo, as expected from the difference in substrate specificity. Clp caused an increase in vascular permeability when injected intradermally into mice, implying a possible role of Clp in the pathogenesis of clostridial myonecrosis.

Clostridium botulinum group III: a group with dual identity shaped by plasmids, phages and mobile elements

“…This type of cysteine endopeptidase has been found in many clostridia, for example in C. novyi and C. botulinum group I genomes, but in those cases it was located on the chromosome. The α-clostripain from C. perfringens has been suggested to be involved in pathogenesis [27]. Two genes coding for metallo-beta lactamases and one for penicillin amidase were also found in this plasmid; these may contribute to resistance to beta-lactam antibiotics.…”

Substrate Profiling and High Resolution Co-complex Crystal Structure of a Secreted C11 Protease Conserved across Commensal Bacteria

“…15–19 Several members of this protease class have been linked to biological and pathogenic processes of the human gut microbiome. For example, the secreted protease fragipain from the commensal microbe Bacteroides fragilis activates the B. fragilis toxin (BFT, fragilysin) and promotes anaerobic sepsis in mice.…”

Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens

“…3A). On the other hand, there was no significant difference between these four strains in the levels of other major secretory proteins, such as CLP (heavy and light chains [11]), ColA, PFO, and 190-kDa hypothetical protein CPE1281 (10), which was identified by N-terminal amino acid sequencing. This indicates that HN1301 is disrupted properly and also suggests that the IF system does not affect the productivity of secretory proteins except for a disrupted one.…”

“…Furthermore, strain 13, which is used in our system, is an extremely poorly sporulating strain with mutation of the master sporulation regulatory gene and hence is suitable in terms of environmental biohazard risk (20). Our system has so far been successfully applied to NanI sialidase (37), ColH collagenase (39), and clostripain-like protease (11).…”