Characterization of two glycosyl hydrolases, putative prophage endolysins, that target Clostridium perfringens.

Clostridium perfringens, a spore-forming anaerobic bacterium, causes food poisoning and gas gangrene in humans and is an agent of necrotizing enteritis in poultry, swine and cattle. Endolysins are peptidoglycan hydrolases from bacteriophage that degrade the bacterial host cell wall causing lysis and thus harbor antimicrobial therapy potential. The genes for the PlyCP10 and PlyCP41 endolysins were found in prophage regions of the genomes from C. perfringens strains Cp10 and Cp41, respectively. The gene for PlyCP10 encodes a protein of 351 amino acids, while the gene for PlyCP41 encodes a protein of 335 amino acids. Both proteins harbor predicted glycosyl hydrolase domains. Recombinant PlyCP10 and PlyCP41 were expressed in E. coli with C-terminal His-tags, purified by nickel chromatography and characterized in vitro. PlyCP10 activity was greatest at pH 6.0, and between 50 and 100 mM NaCl. PlyCP41 activity was greatest between pH 6.5 and 7.0, and at 50 mM NaCl, with retention of activity as high as 600 mM NaCl. PlyCP10 lost most of its activity above 42°C, whereas PlyCP41 survived at 50°C for 30 min and still retained >60% activity. Both enzymes had lytic activity against 75 C. perfringens strains (isolates from poultry, swine and cattle) suggesting therapeutic potential.

The endolysin from the Enterococcus faecalis bacteriophage VD13 and conditions stimulating its lytic activity.

Bacteriophages produce endolysins (peptidoglycan hydrolases) to lyse the host cell from within and release nascent bacteriophage particles. Recombinant endolysins can lyse Gram-positive bacteria when added exogenously. As a potential alternative antimicrobial, we cloned and expressed the enterococcal VD13 bacteriophage endolysin. VD13 endolysin has a CHAP catalytic domain with 92% identity with the bacteriophage IME-EF1 endolysin. The predicted size of VD13 endolysin is ∼27 kDa as verified by SDS-PAGE. The VD13 endolysin lyses Enterococcus faecalis strains, but not E. faecium or other non-enterococci. VD13 endolysin has activity from pH 4 to pH 8, with peak activity at pH 5, and exhibits greater activity in the presence of calcium. Optimum activity at pH 5 occurs in the absence of NaCl. VD13 endolysin, in ammonium acetate (C2H3O2NH4) calcium chloride (CaCl2) buffer pH 5, is stimulated to higher activity upon heating at temperatures up to 65°C for 30 min, whereas activity is lost upon heating to 42°C, in pH 7 buffer.