假结核耶尔森氏菌中Phd-Doc毒素-抗毒素系统的功能鉴定

Functional characterization of Phd-Doc toxin-antitoxin system in Yersinia pseudotuberculosis

摘要:

[背景] 毒素-抗毒素系统在微生物体内广泛存在，在微生物对抗外界不良环境方面发挥重要作用。[目的] 以模式细菌假结核耶尔森氏菌（Yersinia pseudotuberculosis，Yptb）为材料，探究其编码的Phd-Doc毒素-抗毒素系统的作用机制和生物学功能。[方法] 通过生物信息学方法预测Yptb中编码的Phd-Doc毒素-抗毒素系统，通过毒性分析、基因表达分析及蛋白相互作用对其进行鉴定；通过抗生素胁迫、氧胁迫、生物被膜形成等实验研究Phd-Doc在体内发挥的生物学功能。[结果] 生物信息学分析鉴定出一对Phd-Doc毒素-抗毒素系统，发现二者共转录且相互作用；毒素蛋白Doc能够引起大肠杆菌形态发生变化并抑制其生长，抗毒素蛋白Phd能中和Doc的毒性；Phd-Doc毒素-抗毒素系统具有自调控抑制效应；phd-doc的缺失对Yptb自身的生长无影响，而且毒素蛋白Doc在野生型Yptb内过表达并未显示毒性；phd-doc在转录水平上响应了抗生素胁迫和氧胁迫，其中，对氯霉素胁迫最为敏感，但并不影响Yptb的生长；同时，Phd-Doc能够影响Yptb的生物被膜形成能力。[结论] Yptb中Phd-Doc毒素-抗毒素系统的功能鉴定对于更好地了解在多变的外部环境下微生物的定殖和响应机制具有重要意义。

关键词:毒素-抗毒素系统;Phd-Doc;自体调控;环境胁迫;生物被膜

Abstract:

[Background] The toxin-antitoxin system is widespread in microorganisms and plays an important role in their defense against adverse environments. [Objective] we used Yersinia pseudotuberculosis (Yptb) as the meterial to investigate the mechanism and biological functions of the Phd-Doc toxin-antitoxin system. [Methods] The Phd-Doc toxin-antitoxin system encoded in Yptb was identified by bioinformatics, and subsequently confirmed by toxicity experiment, gene expression analysis and protein interaction assay. To study the biological function of Phd-Doc toxin-antitoxin system in Yptb, the differences between the wild type strains and mutant strains were compared through antibiotic stressing, oxygen stressing and biofilm formation experiments. [Results] Bioinformatics analysis showed that a pair of Phd-Doc toxin-antitoxin system is present in Yptb, and subsequent experiments confirmed that these proteins are co-transcripted and display high affinity. The cell morphology was changed and cell growth was inhibited when the Doc toxin protein expressed in Escherichia coli, and these changes were rescued by the antitoxin protein Phd. The transcription level of phd-doc was significantly up-regulated under stress conditions, especially under the chloramphenicol stress. Deletion of phd-doc significantly affected the biofilm forming ability of Yptb but did not influence the cell growth. [Conclusion] The functional identification of the Phd-Doc toxin antitoxin system in Yptb is of great significance for a better understanding of the colonization and response mechanism of microorganisms under the changing external environment.

Key words:toxin-antitoxin system;Phd-Doc;autoregulation;environmental stress;biofilm