Abstract

Staphylococcus aureus is a major cause of infection in humans and animals, causing a wide variety of diseases, from local inflammations to fatal sepsis. The bacterium is commonly multi-drug resistant and thus many front-line antibiotics have been rendered ineffective for treating such infections. Research on murein/peptidoglycan hydrolases, derived from bacterial viruses (bacteriophages), has demonstrated that such proteins are attractive candidates for development as novel antibacterial agents for combatting Gram-positive pathogens. Here we review the research produced to-date on the bacteriophage-derived CHAP_K murein peptidase. Initially, we sequenced and annotated the genome of anti-staphylococcal bacteriophage K and cloned the gene for the bacteriophage endolysin, a murein hydrolase which plays a role in cell killing during the bacteriophage life cycle. An highly active domain of the enzyme, a cysteine, histidine-dependent amido hydrolase/peptidase (CHAP_K), was cloned, overexpressed in E. coli and purified. This CHAP_K enzyme was demonstrated to rapidly lyse several strains of methicillin resistant S. aureus and both disrupted and prevented the formation of a staphylococcal biofilm. The staphylolytic activity of the peptidase was demonstrated in vivo using a mouse model, without adverse effects on the animals. The crystal structure of the enzyme was elucidated, revealing a calcium ion close to the active site. Site-directed mutagenesis indicated that this calcium ion is involved in the catalytic mechanism of the enzyme. The crystal structure of this enzyme is a valuable source of information for efficient engineering of this and similar CHAP-domain-containing proteins. Overall, the data collected to date on CHAP_K has demonstrated its strong potential as a novel therapeutic candidate for treatment of staphylococcal infections and has provided us with insight into the fundamental enzymatic mechanisms of CHAP domaincontaining peptidoglycan hydrolases.

Keywords: Bacteriophage, CHAP Domain, Endolysin, Murein/peptidoglycan hydrolase, Staphylococcus aureus.

Graphical Abstract