假结核耶尔森氏菌硫氧还蛋白系统相关基因参与抗氧化胁迫的机制

Role of the thioredoxin system in response to oxidative stress in Yersinia pseudotuberculosis

摘要:

【背景】硫氧还蛋白系统是细菌重要的氧化还原状态维持系统。假结核耶尔森氏菌(Yersinia pseudotuberculosis) YPⅢ是肠道常见的机会致病菌，在YPⅢ中进行过氧化氢胁迫下的转录组测序，结果发现硫氧还蛋白系统的相关基因有所上调，可能参与细菌氧化胁迫的应答。【目的】研究假结核耶尔森氏菌中硫氧还蛋白系统应答氧化胁迫的方式。【方法】利用同源重组方法构建硫氧还蛋白系统缺失突变株(ΔtrxA、ΔtrxC和ΔtrxR)，研究假结核耶尔森氏菌在过氧化氢胁迫下的生长，测定过氧化氢对细菌的最低抑菌浓度、过氧化氢清除能力，以及过氧化氢处理下的存活率等。同时，通过lacZ报告基因系统对硫氧还蛋白系统的表达调控进行探究。【结果】构建硫氧还蛋白系统缺失突变株，发现缺失trxA后显著影响了假结核耶尔森氏菌的抗氧化胁迫能力，因此TrxA是硫氧还蛋白系统中应答氧化胁迫的主要蛋白；在ΔtrxA中过氧化氢酶的表达上调，但不足以保护细菌及时修复受损的生物大分子；启动子活性测定及实时荧光定量PCR (real time quantitative PCR, RT-qPCR)实验结果表明trxC和trxR的表达可能受到细菌氧化胁迫转录调控因子OxyR的影响。【结论】硫氧还蛋白系统作为细菌内部还原环境的维持系统，在OxyR的调控下参与氧化胁迫应答，并发挥了重要的作用。

Abstract:

[Background] The thioredoxin system is critical for maintaining the redox state in bacteria. Yersinia pseudotuberculosis YPⅢ is a common opportunistic pathogen in the gut. The result of transcriptome sequencing under hydrogen peroxide stress in YPⅢ showed that thioredoxin system related genes are up-regulated, indicating the participation in oxidative stress response.[Objective] Tostudy the mechanism of the thioredoxin system in response to oxidative stress in Yersinia pseudotuberculosis. [Methods] Mutants ΔtrxA, ΔtrxC, and ΔtrxR were constructed by homologous recombination. The mutants were then characterized in terms of the growth curve with or without H₂O₂, minimal inhibition concentration of H₂O₂, ability to degrade H₂O₂, and survival rates under the treatment with H₂O₂. Furthermore, we employed the lacZ reporter system to explore the regulation of the expression of the thioredoxin system.[Results] Mutants ΔtrxA, ΔtrxC, and ΔtrxR were successfully constructed. Only ΔtrxA showed significant sensitivity to H₂O₂, implying that TrxA might be the primary protein in combating with oxidative stress. Although the expression levels of catalases were up-regulated in ΔtrxA, it was still not enough to allow the bacteria to repair the damaging biomacromolecules. In addition, we found that expression levels of trxC and trxR were under positive control of the oxidative stress regulator OxyR. [Conclusion] The thioredoxin system, as a maintenance system for the intracellular reducing state in Y. pseudotuberculosis, participates in the response to oxidative stress under the regulation of OxyR.