霍乱弧菌Ⅵ型分泌系统的效应蛋白对细菌细胞壁的降解机制

Bacterial cell wall degradation by type VI secretion system effector proteins in Vibrio cholerae

摘要:

【背景】肽聚糖(Peptidoglycan，PG)是细菌细胞壁的重要组成部分，而霍乱弧菌Ⅵ型分泌系统(Type VI Secretion System，T6SS)可以分泌具有肽聚糖水解酶活性的效应蛋白到受体细菌中杀死细胞，这类水解酶的作用机制尚未研究清楚。【目的】通过对细菌细胞壁的PG成分进行研究，建立细胞壁PG成分分析方法，并对霍乱弧菌T6SS分泌的2个破坏细胞壁的效应蛋白TseH和VgrG3的作用机制进行解析。【方法】使用显微镜观察TseH和VgrG3异位表达对宿主细菌生长的影响；纯化大肠杆菌细胞壁，使用透射电子显微镜(Transmission Electron Microscope，TEM)观察提纯的细胞壁形态；使用纯化的TseH和VgrG3分解消化PG，利用超高效液相色谱-飞行时间质谱(Ultra-Performance Liquid Chromatography-Time-of-Flight Mass Spectrometry，UPLC-TOFMS)分析鉴定消化后的产物成分；通过分析结果推导结构。【结果】通过透射电子显微镜观察，发现提纯的PG呈现半透明的薄膜泡状；通过UPLC-TOFMS的分析以及逆向推导，得到了提纯的PG被VgrG3水解酶降解之后的3种主要产物，分别是二糖二肽(Disaccharide，Di)、二糖三肽(Disaccharide Tripeptide，Tri)和二糖四肽(Disaccharide Tetrapeptide，Tetra)。【结论】建立了提纯PG和UPLC-TOFMS分析PG成分的方法，揭示了效应蛋白VgrG3而非TseH可以降解PG多糖链N-乙酰葡糖胺和N-乙酰胞壁酸之间的β(1-4)糖苷键的功能。由于攻击细胞壁的效应蛋白在革兰氏阴性细菌中广泛存在，本研究不仅为鉴定这类重要效应蛋白的功能提供了有效的方法，而且对研究靶向细胞壁的新型抗生素也有重要的指导作用。

Abstract:

[Background] Peptidoglycan (PG) is an important component of the bacterial cell wall. The type VI secretion system (T6SS) can secrete effectors with peptidoglycan hydrolase activities into a neighbor bacterial cell to kill the recipient. However, the enzymatic functions of these effectors have not been fully characterized due to the technical challenges in PG analysis. [Objective] We aim to establish an analytical method using liquid chromatography and mass spectrometry to qualitatively determine the PG-hydrolyzing activities of two Vibrio cholerae effectors TseH and VgrG3. [Methods] The antibacterial effects of TseH and VgrG3 were determined by survival assays and microscopy analysis when ectopically expressed in Escherichia coli. Peptidoglycan was purified from E. coli, and morphologically characterized by transmission electron microscopy (TEM). The ultra-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS) was used to identify the products of peptidoglycan from digestion by TseH or VgrG3. [Results] TEM micrograph showed that the purified peptidoglycan is translucent. Using UPLC-TOFMS to analyze VgrG3-treated PG, we identified three products including disaccharide dipeptide (Di), disaccharide tripeptide (Tri), and disaccharide tetrapeptide (Tetra). [Conclusion] VgrG3, rather than TseH, can digest the β(1-4) covalent bond between N-acetylglucosamine and N-acetylmuramic acid. The established method could facilitate the characterization of other cell-wall targeting antibacterial effectors and chemical compounds.