Evaluation of the CHAPK -SH۳b endolysin effect on different strains of Staphylococcus by plate lysis assay

BACKGROUND AND OBJECTIVESExcessive use and misuse of antibiotics have led to the emergence of bacterial strains with multiple resistance, most of which are resistant to more than one type of antibiotic. The emergence of these resistant bacterial species highlights the need to develop alternative treatments. Meanwhile, bacteriophages (phages) and peptidoglycan-degrading enzymes derived from them (endolysins) have shown promising results as an alternative to antibiotics. Endolysins have a fast and unique action, high specificity to kill pathogens, low probability of developing bacterial resistance, and a distinct proteinaceous nature.MATERIALS AND METHODSIn this project, A two-domain chimera recombinant protein composed of the CHAPK domain of endolysin LysK and SH۳b domain of lysostaphin was used. The effect of CHAPK -SH۳b on different serotypes of Staphylococcus aureus(sensitive and resistant to methicillin ) and other strains of Staphylococcus such as Staphylococcus chromogenes, Staphylococcus cohnii, Staphylococcus epidermidis and… was assessed with plate lysis assay. In brief, ۱۰ μL of diluted endolysin was spotted on a freshly prepared bacterial lawn on TSA plates. Spotted plates were air-dried in a laminar flow hood and incubated overnight at ۳۷°C. Cleared spots were digitally photographed within ۲۰ h of plating the cells.RESULTS AND DISCUSSIONCHAPK -SH۳b not only displayed effective lytic activity against all tested methicillin-sensitive staphylococcal strains but was also effective on methicillin-resistant staphylococcus aureus. A difference in lytic ability was observed with different staphylococcal strains, possibly reflecting differences in the cell wall composition between strains. However, other gram-positive bacteria from different genera, including Bacillus cereus and Enterococcus faecalis, were unaffected by CHAPK -SH۳b, suggesting that CHAPK -SH۳b is specific to the genus Staphylococcus.CONCLUSIONWe reported a unique chimeric endolysin with robust lytic activity and an extended-spectrum host range against multiple staphylococcus species in vitro. The SH۳b cell-binding domain of lysostaphin possesses an unusual binding mechanism that allows a synergistic and structurally dynamic recognition of S. aureus peptidoglycan, as the pentaglycine cross-bridge and the peptide stem are recognized by two independent binding sites located on opposite sides of the SH۳b domain. This fact suggests that the lysostaphin SH۳b cell-binding domain facilitates the catalytic domain to find its target bonds within the peptidoglycan, regardless of the presence of modifications in the matured peptidoglycan.

کلیدواژه ها:

Histidine dependent Amidohydrolase Peptidase (CHAP) ، Chimeric protein ، plate lysis assay ، Endolysin ، staphylococcus aureus ، staphylococcal strains