Bacteriophage-derived endolysin enzymes play a critical role in disintegration of the host bacterial cell wall and hence have gained considerable attention as possible therapeutics for the treatment of drug-resistant infections. Endolysins can target both dividing and non-dividing cells and given the vital role peptidoglycan plays in bacterial survival, bacteria are less likely to modify it even if continuously exposed to lysins. Hence, probability of bacteria developing resistance to lysins appear bleak. Endolysins from mycobacteriophages offer great potential as alternative therapeutics for the drug-resistant TB. However, considering that a large number of mycobacteriophages have been discovered so far, the information on endolysins come from only a few mycobacteriophages. In this study, we report the structural and functional characterization of endolysins (LysinA and LysinB) encoded by mycobacteriophage PDRPxv which belongs to B1 sub cluster. On in silico analysis, we found LysinA to be a modular protein having peptidase domain at the N-terminal (104 aa), a central amidase domain (174 aa) and the peptidoglycan binding domain (62 aa) at the C-terminal. Additionally, ‘H-X-H’, which is a conserved motif and characteristic of peptidase domains, and the conserved residues His-His-Asp, which are characteristic of amidase domain were also observed. In LysinB enzyme, a single α/β hydrolase domain having a catalytic triad (Ser-Asp-His) and G-X-S-X-G motif, which are characteristic of the serine esterase enzymes were predicted to be present. Both the enzymes were purified as recombinant proteins and their antimycobacterial activity against M. smegmatis was demonstrated through turbidimetric experiments and biochemical assay. Interesting observation in this study is the secretory nature of LysinA evident by its periplasmic expression in E.coli , which might explain the ability of PDRPxv to lyse the bacterial host in the absence of transmembrane Holin protein.

中文翻译：

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来自分枝杆菌噬菌体 PDRPxv 的细胞内溶素的功能表征

噬菌体衍生的内溶素酶在宿主细菌细胞壁的分解中起关键作用，因此作为治疗耐药性感染的可能疗法受到了相当多的关注。内溶素可以靶向分裂细胞和非分裂细胞，鉴于肽聚糖在细菌存活中的重要作用，即使持续暴露于溶素，细菌也不太可能对其进行修饰。因此，细菌对溶素产生抗性的可能性似乎很渺茫。来自分枝杆菌噬菌体的内溶素作为耐药结核病的替代疗法提供了巨大的潜力。然而，考虑到迄今为止已经发现了大量的分枝杆菌噬菌体，关于内溶素的信息仅来自少数分枝杆菌噬菌体。在这项研究中，我们报告了由属于 B1 亚群的分枝杆菌噬菌体 PDRPxv 编码的内溶素（LysinA 和 LysinB）的结构和功能表征。在计算机分析中，我们发现 LysinA 是一种模块化蛋白质，在 N 端具有肽酶结构域 (104 aa)，在 C 端具有中央酰胺酶结构域 (174 aa) 和肽聚糖结合结构域 (62 aa)。此外，还观察到“HX-H”，它是肽酶域的保守基序和特征，以及保守的残基 His-His-Asp，这是酰胺酶域的特征。在 LysinB 酶中，预测存在具有催化三联体 (Ser-Asp-His) 和 GXSXG 基序的单个 α/β 水解酶结构域，它们是丝氨酸酯酶的特征。这两种酶都被纯化为重组蛋白，并且通过比浊实验和生化分析证明了它们对耻垢分枝杆菌的抗分枝杆菌活性。本研究中有趣的观察结果是 LysinA 在大肠杆菌中的周质表达明显的分泌性质，这可能解释了 PDRPxv 在没有跨膜 Holin 蛋白的情况下裂解细菌宿主的能力。