Biological function identification of phage holin Hol-4086 and treatment of Staphylococcus aureus infection

Chenyu Zheng ^1,2 Yaxin Di ¹ Yingchun Wang ¹ Huyang Zhao ^1,2 Ruichong Wang ³ Guiwei Li ⁴ Xiaona Wang ^1,2 Zhifu San ¹ Yanping Jiang ^1,2 Wen Cui ^1,2 Jiaxuan Li ^1,2 Li Wang ^1,2 Xinyuan Qiao ^1,2*

• ¹ Northeast Agricultural University, Harbin, China
• ² Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Northeast Agricultural University, Harbin, Heilongjiang Province, China
• ³ Heilongjiang Center for Disease Control and Prevention, Harbin, Heilongjiang Province, China
• ⁴ Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang Province, China

The treatment of infections caused by drug-resistant Staphylococcus aureus has become increasingly difficult. In this study, the complete genome of phage 4086-1 against S. aureus was sequenced and shown to be 17960 bp in size, with a GC content of 29.14%. Phylogenetic analysis demonstrated that phage 4086-1 exhibited a close relationship with the Staphylococcus phages SLPW, JPL-50, and LSA2366. BLAST analysis indicated that ORF12 of phage 4086-1 (termed Hol-4086) shares high identity with other reported phage-associated holins. Hol-4086 consists of 140 amino acids and exhibits high sequence identity with some members of the phage_holin_4_1 superfamily (18-125 amino acids). Hol-4086 was then expressed in Escherichia coli and detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting. The results of a spot test demonstrated that Hol-4086 had substantial bacteriostatic effects on the host bacteria. S. aureus cells were exposed to Hol-4086 and observed using transmission electron microscopy and scanning electron microscopy.Bacterial cells treated with Hol-4086 showed ultrastructural and morphological changes. The detection of biofilm activity showed that Hol-4086 effectively inhibited and removed S. aureus biofilms. In vivo, treatment with Hol-4086 significantly reduced the number of bacteria, relieved inflammatory responses, and alleviated pathological changes in the organs of infected mice 48 h after treatment. These results demonstrate that Hol-4086 exhibits promising antibacterial potential as an alternative therapy for the treatment of infections caused by S. aureus.