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首页> 外文期刊>Applied Microbiology >Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C
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Characterization of Lytic Enzyme Open Reading Frame 9 (ORF9) Derived from Enterococcus faecalis Bacteriophage φEF24C

机译：粪肠球菌噬菌体φEF24C衍生的裂解酶开放阅读框9（ORF9）的表征

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摘要

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.
机译：在针对革兰氏阳性细菌（如金黄色葡萄球菌，单核细胞增生性李斯特菌和粪肠球菌）的噬菌体（噬菌体）疗法中，由于其极强的毒力和广泛的宿主谱系，通常采用SPO1类病毒属。噬菌体ΦEF24C是一种感染粪便大肠杆菌的类SPO1病毒，以前已被表征为治疗性噬菌体候选物。除了噬菌体本身，噬菌体内溶素也被认为是有效的抗菌剂。在这项研究中，对粪肠球菌噬菌体ΦEF24C的假定的溶血素基因（orf9）进行了计算机分析，并使用重组体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-乙酰基村酰胺基-1-丙氨酸酰胺酶。其次，通过酶谱法，肽聚糖浊度的降低，活菌数的降低以及ORF9处理的细胞的形态分析证实了ORF9对粪肠球菌的杀菌活性。第三，与生物信息学预测的Zn〜（2+）离子需求相反，ORF9似乎不需要其Zn〜（2+）离子。第四，裂解谱从粪肠球菌菌株的97.1％（35种菌株中，包括万古霉素抗性菌株中的34种）到粪肠球菌菌株的60％（10种菌株中的6种）。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-后源衰变（PSD）分析确认了ORF9的N-乙酰基村酰-1-丙氨酸酰胺酶活性。最后，使用N或C末端缺失的ORF9突变体的功能分析表明，完整的ORF9分子对其活性至关重要。这些结果表明ORF9是噬菌体φEF24C的内溶素，并且可以是抗生素的治疗选择。

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