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

Hiroshi Sekiya,Hirofumi Nariya,Toshi Shimamoto,Eiji Tamai,Maho Okada,Tadashi Shimamoto

doi:10.3390/antibiotics10030245

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.

Highlights

The number of drug-resistant bacteria is increasing worldwide, and the development of new antibacterial drugs is on the decline, a combination that represents a major challenge for the international medical community [1,2,3]
Amino acid sequence alignment analysis suggested that Tyr51 is involved in the catalytic reaction [36] and that His26, His123, and Cys131 are involved in zinc binding, since the amino acids involved in zinc binding in zinc-dependent amidases such as T7 lysozyme and AmiD are conserved in Psa (Figure S1)
The bands of Psa-his, PsaY51F-his, and PsaBD-his disappeared in this cell binding assay, revealing that these proteins are able to bind to C. perfringens. These results demonstrated that the N-terminal domain is the catalytic domain (CD) and that Tyr51 is involved in the catalytic reaction, while the C-terminal domain is the cell wall-binding domain (CBD), without which the CD does not have lytic activity in aqueous solution

Summary

Introduction

The number of drug-resistant bacteria is increasing worldwide, and the development of new antibacterial drugs is on the decline, a combination that represents a major challenge for the international medical community [1,2,3]. Lytic enzymes kill bacteria by degrading the peptidoglycan mesh of the cell wall. Lytic enzymes are composed of a catalytic domain (CD) that hydrolyzes specific sites within peptidoglycan and a cell wall-binding domain (CBD) that recognizes the bacterial cell wall [14]. Two well-known lytic enzymes are endolysin and autolysin. Endolysin is a phage-derived lytic enzyme that is expressed in the final stages of phage infection and is transported to the outside of the infected cell by phage-encoded holins. Genes encoding endolysins are present in dual lysis gene cassettes in association with holin genes [16]. The released endolysin promotes the release of progeny phages by hydrolyzing cell wall peptidoglycan [17,18]. The lytic activity of endolysins generally is species specific.

Methods

Results

Conclusion