Journal
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
Volume 7, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fcimb.2017.00290
Keywords
Staphylococcus aureus; endolysin; antibacterial agent; CHAP-amidisedomain; in silico analysis; antibiotic resistant; synergistic
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Funding
- Kashan University of Medical Sciences, Kashan, Iran [94127]
- Iran National Science Foundation [95821732]
- Deputy of Research and Technology, Ministry of Health and Medical Education of Iran
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Cysteine/ phistidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were previously reported to show lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). In the current study, the in silico design and analysis of chimeric CHAP-amidase model was applied to enhance the stability and solubility of protein, which was achieved through improving the properties of primary, secondary and tertiary structures. The coding gene sequence of the chimeric CHAP-amidase was synthesized and subcloned into the pET-22(+) expression vector, and the recombinant protein was expressed in E. coli BL21 (DE3) strain. Subsequent affinity-based purification yielded similar to 12mg soluble protein per liter of E. coli culture. Statistical analysis indicated that concentrations of >= 1 mu g/mL of the purified protein have significant antibacterial activity against S. aureus MRSA(252) cells. The engineered chimeric CHAP-amidase exhibited 3.2 log reduction of MRSA(252) cell counts at the concentration of 10 mu g/mL. A synergistic interaction between CHAP-amidase and vancomycin was detected by using checkerboard assay and calculating the fractional inhibitory concentration (FIC) index. This synergistic effect was shown by 8-fold reduction in the minimum inhibitory concentration of vancomycin. The chimeric CHAP-amidase displayed strong antibacterial activity against S. aureus, S. epidermidis, and enterococcus. However, it did not indicate any significant antibacterial activity against E. coli and Lactococcus lactis. Taken together, these findings suggest that our chimeric CHAP-amidase might represent potential to be used for the development of efficient antibacterial therapies targeting MRSA and certain Gram-positive bacteria.
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