单核细胞增多性李斯特菌谷胱甘肽合成酶调控鞭毛形成研究

Regulatory role of Listeria monocytogenes glutathione synthetase in bacterial motility and flagellar production

摘要:

[目的] 本试验旨在构建单核细胞增多性李斯特菌谷胱甘肽合成酶基因gshF缺失株和互补株并研究其在细菌运动和鞭毛形成中的调控作用。[方法] 采用同源重组的方法构建得到gshF缺失株后，测定野生株及缺失株的运动性和体外生长能力；同时利用荧光定量PCR方法检测gshF缺失株中鞭毛相关基因转录水平的变化。[结果] 缺失gshF后细菌在体外培养基中的生长能力未受影响，但缺失株的运动性及鞭毛形成能力显著降低。此外，缺失株中鞭毛形成重要调控基因gmaR以及鞭毛结构元件基因flaA的转录水平显著低于野生株，而其他鞭毛相关基因的转录水平变化不明显。[结论] 研究首次表明单增李斯特菌谷胱甘肽合成酶通过调控鞭毛重要基因的转录进而影响细菌的运动性和鞭毛形成，研究有助于深入理解重要食源性胞内菌通过精确调控鞭毛形成以适应外界和宿主环境的分子机制。

Abstract:

[Objective] The foodborne pathogen Listeria monocytogenes is well-adapted outside of the environmental conditions and inside of the host niche by delicately controlling flagellar production. We here aimed to explore the regulatory roles of L. monocytogenes glutathione synthetase (encoded by gshF) in bacterial motility and flagella production.[Methods] The gene knock-out mutant and complemented strains (ΔgshF and CΔgshF) were constructed and then subjected to in vitro bacterial growth and motility assays compared with the wild-type strain EGD-e. In addition, transcriptional changes of the flagella-associated genes were determined for the wild-type and gshF mutant strains using the Real-time Quantitative Reverse Transcription PCR (qRT-PCR) method.[Results] The data showed that in vitro growth ability was not significantly affected by deletion of gshF while the swarming ability of ΔgshF was markedly impaired. More importantly, the transcriptional levels of the flagellar-associated factors GmaR, a protein thermometer that controls temperature-dependent transcription of flagellar genes, and FlaA, encoding flagellin, were most significantly down-regulated at 30℃ in the absence of gshF.[Conclusion] We in this study showed that the glutathione synthetase of L. monocytogenes plays a vital role in controlling transcription of flagellar genes and contributes to bacterial swarming motility and flagellar production. These data would be helpful to better understand the regulatory mechanisms employed by this intracellular pathogen to favor environmental adaption and host infection.