Hfq对霍乱弧菌环境适应能力的影响及相关基因的转录调控  

Involvement of Hfq in stress response and transcriptional regulation of virulence genes and stress-responsive genes in Vibrio cholera

作　　者：陈秀琴[1] 闫志勇[1] 李杰[2] 王瑞白[2] 闫梅英[2] 阚飙[2] 高鹤[2] 

机构地区：[1]青岛大学医学院微生物学教研室,266071 [2]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,北京102206

出　　处：《中华微生物学和免疫学杂志》2013年第4期241-246,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（30830008）;中国疾病预防控制中心青年科研基金（2012A103）

摘　　要：目的通过表型实验分析Hfq蛋白对霍乱弧菌环境适应能力的影响，并通过分子实验验证Hfq对环境生存及致病相关基因转录的调控。方法基于自杀质粒缺失霍乱弧菌的hfq基因，并构建相应回补株；通过表型实验，比较霍乱弧菌野生株和hfq突变株在细菌运动能力、生物膜形成、外膜抗性和氧化应激能力上的差异；应用逆转录荧光定量PCR（RT-qPCR）技术对Hfq可能调控的相关基因的转录水平进行比较。结果成功构建霍乱弧菌hfq缺失株及回补株，表型实验表明hfq突变株的菌株游动性降低，生物膜形成能力增强，对十二烷基磺酸钠（SDS）和庆大霉素的耐受性显著下降，并且与野生株相比，hfq突变株对H2O2刺激更为敏感。RT-qPCR实验证明Hfq对霍乱弧菌鞭毛合成、胞外多糖合成、外膜蛋白以及氧化应激相关基因的转录有调控作用。结论Hfq蛋白作为RNA伴侣分子，能够通过调控一些生存及致病相关基因的转录进而影响霍乱弧菌的生物膜形成、氧化应激反应、抗生素抗性等能力，表明Hfq可能与其他调控子一起，在霍乱弧菌的生存、代谢以及致病因子转录的协调控制上起关键作用。Objective To characterize Hfq-dependent phenotypes in stress response and to dissect Hfq-dependent transcription of virulence genes and stress-responsive genes in Vibrio cholera. Methods The hfq null mutant strain （Ahfq） and the complemented mutant strain （Ahfq/pUC18-hfq） were constructed from the wild-type Vibrio cholera. Comparisons on the motility, biofilm formation, growth under various oxygen-supplying conditions, outer membrane resistance, and sensitivity to oxidative stress were analyzed between the wild type strain and the mutant strains. Reverse-transcript fluorescence quantitative PCR （RT- qPCR） was used to determine the transcriptional levels of target genes in the above mentioned strains. Resuits Ahfq and Ahfq/pUC18-hfq strains were successfully constructed. The motility, outer membrane resistance and sensitivity to oxidative stress were reduced, but biofilm formation was enhanced in the hfq null mutant strain. RT-qPCR testified that Hfq had regulation effects on gene transcription for forming falagellum, extraeellular polysaccharide, outer membrane protein and oxidative stress in Vibrio cholera. Conclusion As a RNA chaperone, Hfq could affect Vibrio cholera in its biofilm formation, resistance to oxidative stress and antibiotics resistance through regulating the transcription of multiple metabolic genes and virulence genes, which indicates that Hfq, combined with other regulators, may play a key role in the complex regulation of metabolic genes and virulence genes.

关 键 词：霍乱弧菌 HFQ 环境适应 转录调控 

分 类 号：R[医药卫生]