| Staphylococcus aureus（S. aureus） is an important Gram-positive zoonotic pathogen. It can cause a variety of local and systemic infections. Lytic bacteriophages are viruses that lysis specific pathogen quickly at unique mechanism of action. It has shown great potential application to treat drug-resistant bacteria infection. In this study, the S. aureus phage GH15 lysis system were studied. It has revealed three-dimensional structure, mechanism of lytic enzymes, on the basis of focusing on holin. The function and activity of Hol GH15 gene were determined and effective of disruption on MRSA biofilms by Lys GH15 and phage GH15 were studied.First of all, sequence alignment and bioinformatics analysis to predict the holin gene of bacteriophage GH15. Holin of phage GH15 was located in lysis module and next to the Lys GH15 gene. Hol GH15 have membrane protein of two transmembranes area, its N- and C-terminals are distributed in the cytoplasm, possessesing characteristics of holin II class. Hol GH15 conserved regions of 13 to 95 amino acids exhibits high sequence identity with some members that belong to the phageholin1 superfamily. The amino acid sequence of Hol GH15 was also compared with other published putative holins of Gram-positive phages, such as the Staphylococcus, Streptococcus suis and Lactobacillus fermentum phages.The sequence alignment demonstrated that Hol GH15 showed high level of identity with the putative holins derived from Staphylococcus phage K（95% identity at the amino acid level）, and other less than 60%.To further investigate, the holin was expressed and purified. In expression process, Hol GH15 caused growth inhibition of host E. coli cells. Additionally, Hol GH15 was localized on the cell membrane, sensitive to energy poisoning with DNP.TMDs are major components of holins and play an important role in exerting the active functions of this type of protein. Mutation analysis indicated that both TMD1 and TMD2 of Hol GH15 are indispensable for the function of the full-length protein.We found that exogenous Hol GH15 displayed antibacterial activity against S. aureus. Bacterial cells were most susceptible to Hol GH15 at 37℃. The greatest antimicrobial activity of Hol GH15 was detected at p H 5.2. Interestingly, Hol GH15 also displayed antibacterial activity against other species, including both Gram-negative and Gram-positive species, such as Bacillus. subtilis, E. coli, Klebsiella. pneumoniae and Pseudomonas. aeruginosa. The minimum bactericidal activity（MBC） assay showed that Hol GH15 showed bactericidal activity at L.monocytogenes, the other is antibacterial activity. Hol GH15 had no haemolytic activity. Hol GH15 displayed antibacterial and bactericidal activity by changing the membrane permeability of the bacterial cell.Bacterial biofilms are crucial to the pathogenesis of persistent infection caused by MRSA, phage GH15 and Lys GH15 showed a broad host range and strong lytic activity to MRSA. In this study, the MRSA biofilms were cultured in a 24-well plate. The result showed that MRSA strains have different degree capabilities to form biofilms.The biofilms were disrupted by phage GH15 and Lys GH15 respectively. At the same time, phage GH15 can prevent biofilm formation. Phage GH15 can keep stable activity in nonionic detergents, 10 mmol EDTA, distilled water and 10% serum, that exerted more powerful ability to disrupt biofilm combined with 1% Triton X-100. Lys GH15 can disrupt almost all of the MRSA biofilm at 10 μg / m L in 1h.Because β-lactam antibiotics can inhibit the formation of the bacterial cell wall, Hol GH15 change the bacterial cell membrane permeability. Hol GH15 and β-lactam antibiotics have enhanced synergistic antibacterial activity. Hol GH15 was able to efficiently inhibit and kill L. monocytogenes at 4°C. Thus, Hol GH15 has a application prospect.In conclusion, phage GH15, Lys GH15 and Hol GH15 exhibits great potential as a candidate novel antibacterial agents to control S. aureus biofilms, L. monocytogenes and associated infections. |
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