解码分枝杆菌噬菌体 D29 Holin 的分子特性为 Holin 工程提供了见解,Journal of Virology

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解码分枝杆菌噬菌体 D29 Holin 的分子特性为 Holin 工程提供了见解
Journal of Virology ( IF 4.0 ) Pub Date : 2021-04-26 , DOI: 10.1128/jvi.02173-20
Varun Rakeshbhai Bavda ₁ , Aditi Yadav ₁ , Vikas Jain ₂

Affiliation

Holins 是噬菌体编码的小跨膜蛋白，通过在感染后的特定时间在宿主细胞膜上形成非特异性孔来确定噬菌体感染周期的持续时间。因此，穴蛋白也被称为“蛋白质时钟”。穴蛋白有一个或多个跨膜结构域，以及一个带电的 C 末端区域，虽然在穴蛋白中是保守的，但尚未进行详细检查。在这里，我们描述了分枝杆菌噬菌体 D29 穴蛋白 C 末端区域的分子特性，并研究了其中存在的带电残基和卷曲螺旋 (CC) 结构域的重要性。我们表明 CC 结构域对于穴蛋白介导的有效细菌细胞裂解是必不可少的。我们进一步证明，在 C 端区域中存在的带正电荷和带负电荷的残基中，用前者代替，而不是后者，丝氨酸使穴蛋白无毒。此外，第 59 位和第 79 位氨基酸之间存在的碱性残基对于穴蛋白介导的毒性最为关键。我们还通过复制 C 端区域构建了工程穴蛋白 HolHC。与野生型相比，HolHC 蛋白在两个方面都表现出更高的毒性大肠杆菌和耻垢分枝杆菌，并在表达后迅速杀死这两种细菌。HolHC 的寡聚化特性类似于野生型穴蛋白的寡聚化特性，使我们能够提出 D29 穴蛋白的 C 末端区域决定了寡聚化的时间，而不是程度，从而决定了空穴的形成。这种基于知识的分枝杆菌噬菌体穴蛋白工程将有助于开发新的基于噬菌体的疗法来杀死致病性分枝杆菌，包括结核分枝杆菌。

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Decoding the Molecular Properties of Mycobacteriophage D29 Holin Provides Insights into Holin Engineering

Holins are bacteriophage-encoded small transmembrane proteins that determine the phage infection cycle duration by forming nonspecific holes in the host cell membrane at a specific time postinfection. Thus, holins are also termed “protein clocks.” Holins have one or more transmembrane domains, and a charged C-terminal region, which, although conserved among holins, has not yet been examined in detail. Here, we characterize the molecular properties of the mycobacteriophage D29 holin C-terminal region, and investigate the significance of the charged residues and coiled-coil (CC) domain present therein. We show that the CC domain is indispensable for holin-mediated efficient bacterial cell lysis. We further demonstrate that, out of the positively- and negatively-charged residues present in the C-terminal region, substituting the former, and not the latter, with serine, renders holin nontoxic. Moreover, the basic residues present between the 59th and the 79th amino acids are the most crucial for holin-mediated toxicity. We also constructed an engineered holin, HolHC, by duplicating the C-terminal region. Compared to the wild type, the HolHC protein shows higher toxicity in both Escherichia coli and Mycobacterium smegmatis and causes rapid killing of both bacteria upon expression. An oligomerization property of HolHC similar to that of the wild-type holin allows us to propose that the C-terminal region of the D29 holin determines the timing, and not the extent, of oligomerization and, thereby, hole formation. Such knowledge-based engineering of mycobacteriophage holin will help in developing novel phage-based therapeutics to kill pathogenic mycobacteria, including Mycobacterium tuberculosis.

更新日期：2021-04-26

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