Department of Microbiology and Infection, Faculty of Medicine, Kochi University, Nankoku City, Kochi, Japan.
Appl Environ Microbiol. 2011 Jan;77(2):580-5. doi: 10.1128/AEM.01540-10. Epub 2010 Nov 19.
In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.
在针对革兰氏阳性菌（如金黄色葡萄球菌、单核细胞增生李斯特菌和粪肠球菌）的噬菌体（噬菌体）治疗中，通常使用类似于 SPO1 的病毒属的成员，因为它们具有极强的毒性和广泛的宿主谱。噬菌体 φEF24C 是一种感染粪肠球菌的类似于 SPO1 的病毒，以前被认为是一种有治疗潜力的噬菌体候选物。除了噬菌体本身，噬菌体溶菌素也被认为是一种有效的抗菌剂。在这项研究中，对粪肠球菌噬菌体 φEF24C 的推定溶菌素基因（orf9）进行了计算机分析，并通过重组形式对其活性进行了表征。首先，生物信息学分析预测开放阅读框 9（ORF9）蛋白是 N-乙酰基胞壁酰-L-丙氨酸酰胺酶。其次，通过酶谱、肽聚糖浊度降低、活菌计数减少和 ORF9 处理细胞的形态分析，证实了 ORF9 对粪肠球菌的溶菌和杀菌活性。第三，ORF9 的活性似乎不需要 Zn（2+）离子，这与生物信息学预测的需要 Zn（2+）离子的情况相反。第四，溶菌谱从 35 株粪肠球菌菌株中的 97.1%（34 株，包括耐万古霉素菌株）到 10 株屎肠球菌菌株中的 60%（6 株）。第五，通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和随后的 MALDI-源后降解（PSD）分析，证实了 ORF9 的 N-乙酰基胞壁酰-L-丙氨酸酰胺酶活性。最后，使用 N 或 C 端缺失的 ORF9 突变体进行功能分析表明，完整的 ORF9 分子是其活性所必需的。这些结果表明，ORF9 是噬菌体 φEF24C 的溶菌素，可以作为抗生素的替代疗法。
