摘要/Abstract

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 phoP及其下游基因的转录水平。结果 ·成功构建 30个敲除株。Δ STM14_0739、ΔSTM14_2717和ΔSTM14_1646对数期酸耐受能力较 WT增强，Δ STM14_1878、ΔSTM14_4338和ΔSTM14_1965则较 WT减弱。Δ STM14_0739和ΔSTM14_2717对 HeLa细胞的入侵率降低，Δ STM14_1878和Δ STM14_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 phoP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。
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
Abstract:
Objective · To uncover new transcriptional regulatorsscreening 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 deletedλ-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 detectedrealtime-PCR in the mutants involved in both ATR and cell infection. Results · All 30 deletion mutants were successfully constructed. ΔSTM14_0739, ΔSTM14_2717 and ΔSTM14_1646 showed increased log-phase ATR ability, while ATR abilities of ΔSTM14_4338, ΔSTM14_1965 and ΔSTM14_1878 decreased, compared with WT. ΔSTM14_0739 and ΔSTM14_2717 mutants showed weaker invasion ability in HeLa cells than WT, and ΔSTM14_1878 and ΔSTM14_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.
Key words: Salmonella enterica serovar Typhimurium, transcriptional regulators, acid tolerance response, invasion and replication


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