单增李斯特菌二硫键形成蛋白DsbG介导酸耐受及鞭毛运动性调控

Role of DsbG in acid tolerance and flagella-mediated motility of Listeria monocytogenes

摘要:

【目的】本研究旨在通过构建单增李斯特菌(L isteria monocytogenes)二硫键形成蛋白编码基因dsbG缺失株和回补株，探究该蛋白在酸耐受和鞭毛介导的运动性中发挥的生物学功能。【方法】利用同源重组方法构建单增李斯特菌dsbG缺失株和回补株，比较野生株和突变株在不同pH梯度培养基条件下的生长速率和存活率；通过实时荧光定量PCR方法，比较致死酸应激条件下野生株和缺失株酸耐受基因转录水平。通过半固体培养基、荧光定量PCR方法和鞭毛负染色透射电镜观察，比较野生株和突变株的运动能力、鞭毛相关基因转录水平变化和鞭毛形态差异。【结果】与野生株相比，dsbG缺失株的生长能力和速率差异不显著；在pH 3.5 (盐酸和柠檬酸)培养条件下，存活率显著降低；精氨酸合成途径基因argD和argF转录水平分别下调2.4和3.7倍。同时，dsbG缺失株的运动能力减弱，且鞭毛形成相关的flaA、flgB和flgD等基因转录水平显著下调(分别为29.7、6.7和6.9倍)，鞭毛形成能力减弱。【结论】本研究首次证实了单增李斯特菌二硫键形成蛋白DsbG能感应低pH应激，并形成耐受；证实了DsbG通过调控鞭毛相关基因的转录进而影响细菌的鞭毛形成和运动性。本研究有助于深入了解二硫键形成蛋白家族介导单增李斯特菌环境适应的分子机制，为食源性致病菌的防控提供理论基础。

关键词:单增李斯特菌;二硫键形成蛋白;酸耐受;运动性;鞭毛

Abstract:

[Objective] To investigate the biological roles of the disulfide bond formation protein DsbG in the acid tolerance and flagella-mediated motility of Listeria monocytogenes by constructing the dsbG-deleted and complemented strains. [Methods] The dsbG-deleted and complemented strains were constructed via the homologous recombination method. The growth and survival rates of the strains in the media at different pH gradients were analyzed. The transcriptional levels of the genes involved in acid tolerance were determined by real-time quantitative PCR and compared between the wild-type and mutant strains exposed to acid stress. The motility, transcriptional levels of flagella-associated genes, and flagellar morphology were compared between wild-type and mutant strains by semi-solid culture, real-time quantitative PCR, and transmission electron microscopy, respectively. [Results] The deletion of dsbG did not affect the growth of the bacteria in a medium with neutral pH. However, it significantly reduced the survival rate of bacteria in the media with hydrochloric acid and citric acid (pH 3.5). It down-regulated the transcriptional levels of the genes (argD and argF) in the arginine pathway by 2.4 and 3.7 times, respectively. Moreover, the lack of dsbG impaired the flagellum formation and bacterial motility and down-regulated the transcriptional levels of flagella-associated genes flaA, flgB, and flgD by 29.7, 6.7, and 6.9 times, respectively, compared with the wild-type strain. [Conclusion] This study for the first time, demonstrated that DsbG could sense low pH stress, play a role in the development of acid tolerance, and more importantly, contribute to the flagella-mediated motility of L. monocytogenes via regulating the transcription of flagella-associated genes. This study helps to understand the roles of the disulfide bond formation protein family in the environmental adaptation of L. monocytogenes and provides a theoretical basis for the prevention and control of foodborne pathogens.

Key words:Listeria monocytogenes;disulfide bond formation protein;acid tolerance;motility;flagella