假结核耶尔森氏菌T6SS效应蛋白TseN功能鉴定

Functional characterization of the T6SS effector TseN in Yersinia pseudotuberculosis

摘要:

【背景】 细菌六型分泌系统(type VI secretion system, T6SS)广泛分布于革兰氏阴性细菌，在细菌定殖、环境适应、细菌-宿主互作等过程中发挥重要作用；T6SS对致病菌假结核耶尔森氏菌(Yersinia pseudotuberculosis)的致病性有重要作用。【目的】 探究假结核耶尔森氏菌T6SS基因簇编码的潜在效应蛋白T6SS分泌型效应核酸酶(T6SS secreted effector nuclease, TseN)的生理生化特性及其生物学功能。【方法】 通过生物信息学分析锁定潜在的T6SS效应蛋白TseN及其免疫蛋白T6SS分泌型免疫核酸酶(T6SS secreted immune nuclease, TsiN)；构建相关重组质粒转化大肠杆菌(Escherichia coli)，利用生长曲线测定等方法探究TseN的细菌毒性及TsiN免疫活性；通过细菌双杂交试验验证TseN与TsiN的相互作用；纯化蛋白，通过体外DNA酶活实验明确TseN的DNA酶活特性；利用同源重组等技术构建假结核耶尔森氏菌tseN基因缺失菌株(ΔtseN)，并构建回补菌株[ΔtseN(tseN)]，确证TseN在细菌竞争、抵抗胁迫等方面的功能。【结果】 表达TseN的大肠杆菌生长受到显著抑制，共表达TsiN的菌株生长恢复，并且TsiN通过与TseN直接相互作用来中和其毒性。TseN可以结合DNA，具有Mn²⁺依赖的DNA水解酶活性。相较于野生型菌株，ΔtseN介导细菌种内或种间竞争的能力显著减弱，并且ΔtseN的生物膜形成受到显著影响，面对高盐、低pH等胁迫条件时存活率显著降低，以上情况在回补菌株中得到一定恢复。【结论】 TseN是假结核耶尔森氏菌T6SS分泌的Mn²⁺依赖的DNA水解酶，具有较强的细菌毒性作用；TsiN是其免疫蛋白，保护假结核耶尔森氏菌免受TseN的毒性作用。TseN介导假结核耶尔森氏菌参与细菌竞争，有助于其占据生态位。同时，能促进假结核耶尔森氏菌的生物膜形成、高盐和低pH等胁迫耐受能力。

关键词:假结核耶尔森氏菌;T6SS;效应蛋白;免疫蛋白;功能鉴定

Abstract:

[Background] The type VI secretion system (T6SS) with wide distribution in Gram-negative bacteria plays important roles in bacterial colonization, environmental adaptation, and bacterium-host interactions. T6SS is crucial for the pathogenicity of Yersinia pseudotuberculosis. [Objective] To investigate the physiological and biochemical characteristics and the biological function of the potential effector TseN encoded by the T6SS gene cluster of Y. pseudotuberculosis. [Methods] The bioinformatics analysis was performed to identify the potential T6SS effector TseN and its immune protein TsiN. Recombinant plasmids were constructed and transformed into Escherichia coli, and growth curves were established to evaluate the toxicity of TseN to bacteria and the immune activity of TsiN. The bacterial two-hybrid system was used to verify the interaction between TseN and TsiN. The in vitro DNA nuclease activity assay was employed to confirm the DNA nuclease activity of the purified TseN. Y. pseudotuberculosis mutant with tseN deletion (ΔtseN) was constructed by homologous recombination, and a complemented strain (ΔtseN(tseN)) was generated to examine the roles of TseN in bacterial competition and stress resistance. [Results] The expression of TseN significantly inhibited the growth of E. coli, which was restored in the strain co-expressing TseN and TsiN. The result indicated that TsiN neutralized the toxicity of TseN by direct interaction. TseN could bind to DNA and exhibited Mn²⁺-dependent DNA nuclease activity. Compared with the wild-type strain, ΔtseN showed significantly reduced ability in mediating intra- or inter-species bacterial competition, and its biofilm formation was significantly affected. In addition, ΔtseN showcased decreased survival rate under high salt, low pH, and other stress conditions, with partial recovery observed in the complemented strain. [Conclusion] TseN is a Mn²⁺-dependent DNA nuclease secreted by the T6SS of Y. pseudotuberculosis, with strong toxicity to bacteria. TsiN is the immune protein of TseN, protecting Y. pseudotuberculosis from the toxic effects of TseN. TseN play a role in the bacterial competition of Y. pseudotuberculosis by helping the bacterium occupy ecological niches, promoting biofilm formation, and enhancing tolerance to stress conditions such as high salt and low pH.

Key words:Yersinia pseudotuberculosis;type VI secretion system (T6SS);effector;immune protein;functional characterization