鼠伤寒沙门菌中假定转录调控因子的筛选  

Screening of putative transcriptional regulators in Salmonella enterica serovar Typhimurium

作　　者：刘舒婷 姚玉峰[1] 倪进婧[1] LIU Shu-ting;YAO Yu-feng;NI Jin-jing(Department of Pathogeny Biology,Shanghai Jiao Tong University College of Basic Medical Sciences,Shanghai 200025,China)

机构地区：[1]上海交通大学基础医学院病原生物学教研室,上海200025

出　　处：《上海交通大学学报（医学版）》2018年第10期1174-1180,共7页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(31700121)~~

摘　　要：目的·基因组信息学分析结合分子生物学实验对鼠伤寒沙门菌中假定转录调控因子进行筛选,以期发现新的转录调控因子和调控机制。方法·通过沙门菌基因组信息分析筛选得到30个假定转录调控因子,利用λ-Red同源重组系统敲除各目的基因。酸耐受实验检测各敲除株在对数期酸耐受应答中的差异;细胞感染模型检测各敲除株上皮细胞入侵能力和巨噬细胞内复制能力;利用realtimePCR检测突变株中pho P及其下游基因的转录水平。结果·成功构建30个敲除株。ΔSTM14_0739、ΔSTM14_2717和ΔSTM14_1646对数期酸耐受能力较WT增强,ΔSTM14_1878、ΔSTM14_4338和ΔSTM14_1965则较WT减弱。ΔSTM14_0739和ΔSTM14_2717对He La细胞的入侵率降低,ΔSTM14_1878和ΔSTM14_2717在RAW264.7细胞内的24 h复制率提高。Realtime-PCR结果显示STM14_2717敲除并不影响pho P转录。结论·研究发现了新的转录调控因子,为深入研究沙门菌致病机制提供新的方向。Objective·To uncover new transcriptional regulators by screening putative transcriptional regulators in Salmonella enterica serovar Typhimurium(S.Typhimurium)through genome informatics and molecular biology analyses.Methods·S.Typhimurium genome informatics was analyzed and 30 putative transcriptional regulators were screened out.All candidate genes were deleted byλ-Red system.The log-phase acid tolerance response(ATR)ability was compared between knock-out(KO)strains and wild type(WT)strain.Cell model was used to detect the invasion ability and intracellular ability of KO strains that showed differences in acid stress compared to WT strain.The transcription level of phoP was detected by realtime-PCR in the mutants involved in both ATR and cell infection.Results·All 30 deletion mutants were successfully constructed.ΔSTM14_0739,ΔSTM14_2717 andΔSTM14_1646 showed increased log-phase ATR ability,while ATR abilities ofΔSTM14_4338,ΔSTM14_1965 andΔSTM14_1878 decreased,compared with WT.ΔSTM14_0739 andΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT,andΔSTM14_1878 andΔSTM14_2717 mutants showed stronger replication ability in RAW264.7 cells than WT.Realtime-PCR suggested STM14_2717 deletion had no effect on phoP transcription.Conclusion·This work discovers unknown transcriptional regulators,and provides clues for future research in S.Typhimurium pathogenesis.

关 键 词：鼠伤寒沙门菌 转录调控因子 酸耐受 细胞入侵及胞内复制 

分 类 号：R37[医药卫生—病原生物学]