A Putative Amidase Endolysin Encoded by Clostridium perfringens St13 Exhibits Specific Lytic Activity and Synergizes with the Muramidase Endolysin Psm

Abstract

Clostridium perfringens is an often-harmful intestinal bacterium that causes various diseases ranging from food poisoning to life-threatening fulminant disease. Potential treatments include phage-derived endolysins, a promising family of alternative antimicrobial agents. We surveyed the genome of the C. perfringens st13 strain and identified an endolysin gene, psa, in the phage remnant region. Psa has an N-terminal catalytic domain that is homologous to the amidase_2 domain, and a C-terminal domain of unknown function. psa and gene derivatives encoding various Psa subdomains were cloned and expressed in Escherichia coli as N-terminal histidine-tagged proteins. Purified His-tagged full-length Psa protein (Psa-his) showed C. perfringens-specific lytic activity in turbidity reduction assays. In addition, we demonstrated that the uncharacterized C-terminal domain has cell wall-binding activity. Furthermore, cell wall-binding measurements showed that Psa binding was highly specific to C. perfringens. These results indicated that Psa is an amidase endolysin that specifically lyses C. perfringens; the enzyme’s specificity is highly dependent on the binding of the C-terminal domain. Moreover, Psa was shown to have a synergistic effect with another C. perfringens-specific endolysin, Psm, which is a muramidase that cleaves peptidoglycan at a site distinct from that targeted by Psa. The combination of Psa and Psm may be effective in the treatment and prevention of C. perfringens infections.

Direct Lytic Agents: Novel, Rapidly Acting Potential Antimicrobial Treatment Modalities for Systemic Use in the Era of Rising Antibiotic Resistance

“…Lysins with in vitro bacteriolytic activities against an array of Gram-positive organisms, including staphylococci, streptococci, Clostridium spp., Paenibacillus larvae , Bacillus anthracis , Listeria monocytogenes and others, have been described and reviewed in the literature ( Schuch et al, 2002 ; Nelson et al, 2012 ; Oliveira et al, 2015 ; Fischetti, 2017 ; Haddad Kashani et al, 2018 ; Murray et al, 2021 ; Sekiya et al, 2021 ). A limited number of these have advanced toward the clinic.…”

Battling Enteropathogenic Clostridia: Phage Therapy for Clostridioides difficile and Clostridium perfringens

“…Virtually all endolysins originating from Cp phages exclusively showed lytic activity against Cp. A recent study on the endolysin Psa and its catalytic domain (Psa-catalytic domain) shed light on the governing factors of this specificity ( Sekiya et al, 2021a , b ). The species specificity of Psa lytic activity primarily depended on the binding specificity of the cell wall-binding domain, as also observed for other endolysins ( Zimmer et al, 2002b ).…”

“…In the case of Cp, the combination of endolysins Psa and Psm ( Supplementary Table 1 ) showed a synergistic effect on the lysis of the bacterium. This was due to the fact that both endolysins target different regions in the peptidoglycans ( Sekiya et al, 2021a ). A synergistic killing effect was already demonstrated for two phage-encoded lysins for Streptococcus pneumoniae , which similarly target distinct regions in the cell wall of the host bacterium ( Loeffler and Fischetti, 2003 ).…”

“…However, the in vivo efficacy of such a combination approach remains to be confirmed. Suggested applications of the enzyme mixture included the spraying of areas to treat drug-resistant Cp, pre-mixing into poultry feed against foodborne Cp infections, and enteric capsule preparations for precision targeting to fight intestinal diseases ( Sekiya et al, 2021a ).…”

“…Cp strains st13 and SM101 have fully sequenced genomes and have been the most extensively studied with respect to C. perfringens pathogenicity ( Sekiya et al, 2021a ). Aside from these fully sequenced strains, further comparative genome analysis of 206 publicly available Cp strains’ sequence data indicated five closely-related phylogenetic groups (phylogroups) reflecting different disease involvements ( Abdel-Glil et al, 2021 ), thus demonstrating the large repertoire of potential virulence factors.…”