长链反义RNA调控伤寒沙门菌毒力机制研究

In recent years, many bacterial non-coding RNAs (ncRNAs) have been found, which involved in bacterial responses to environmental stresses and the expressional regulation of virulence genes. By the deep RNA sequencing analysis of transcriptome, we recently found many novel ncRNAs in Salmonella enterica serovar Typhi. Two novel long antisense RNAs (asRNAs) about 700-800nt, AS-RpoH and AS-RseC were also identified by Northern blot assay, which gene locatted in opposites of regulator gene rpoH and rseC. Change of the expression of tow asRNAs in S.enterica serovar Typhi could influence the bacterial invasion ability and the growth rate under oxidative stress, which indicated that both of two asRNAs are highly involved in the virulence of S. enterica serovar Typhi. In this program, we want to analyze the molecular characteristics of AS-RpoH, AS-RseC in S. enterica serovar Typhi, including transcriptional initiating sites, promoter and encoding regions, expression dynamics and expressional regulation mechanisms at environmental stresse conditions. The effect of them on bacterial systemic gene expression also will be investigated by using mutagenesis, high expression and some other advanced techniques on genomics and proteomics, such as genomic microarray and 2DE-MS assay. The mechanisms of regulating expression of target genes, especially Salmonella pathogenic genes by them could be clarified,　as cis- or trans-action. Results of the study may enhance the understanding about networks of the expressional regulation of virulence genes in S. enterica serovar Typhi.

已知许多细菌非编码RNA能参与致病基因的表达调控。最近我们通过对伤寒沙门菌多种环境应激下的转录组深度序列分析，发现了许多新的非编码RNA，用Northern blot验证了调节基因rpoH和rseC反义链分别编码有长约700-800nt的反义RNA（AS-RpoH和AS-RseC），干预其表达，能明显影响细菌侵袭力和氧应激下的生长速度，说明其与细菌的毒力密切相关。为揭示AS-RpoH和AS-RseC参与伤寒沙门菌毒力调控的作用机制，本项目拟详细分析伤寒沙门菌AS-RpoH和AS-RseC的分子特性，包括明确其转录起始和编码区域；分析其在各种环境应激条件下的表达特性及调节机制；通过制备各种基因突变和高表达菌株，结合应用基因组学和蛋白组学技术，系统分析其对细菌毒力相关基因的表达影响，并深入分析其对毒力基因表达调控的顺式或反式作用机制。研究结果将能增进对沙门菌致病基因表达调节网络的认识。

本研究在前期利用高通量测序技术分析伤寒沙门菌转录组基础上，针对新发现的长链非编码RNA开展深入研究，主要包括两种长链非编码RNA：AS-rseC (AsrC), AS-rpoH (AsrH) 以及5'-UTR-malS，利用一系列分子细菌学研究技术和方法，对非编码RNA进行分子特性鉴定、编码基因定位、分子表达和调节、功能和作用机制进行研究，主要结果发现：（1）伤寒沙门菌AsrC基因编码定位于rseC基因编码对侧，全长894nt，环境应激下受RpoE, RpoS, OxyR调控，细菌生长对数中晚期表达较高，主要受RNaseE降解，能通过增加rseC的mRNA的稳定性而促进RseC的表达，间接促进RpoE的表达，从而有利于细菌的动力和侵袭力的提升；（2）伤寒沙门菌AsrH基因编码定位于rpoH基因编码对侧，长度3050－3097nt，覆盖855bp全长的rpoH基因，并包含了yhhK基因，在细菌生长的稳态期表达较高，主要受RNaseIII降解，环境酸应激时通过RpoE下调表达，高表达有利于细菌有酸应激下的生长，基因芯片表达谱分析发现其能促进鞭毛和SPI1相关基因表达，主要通过增加的rpoHmRNA的稳定性而促进RpoH的表达，从而提高细菌的侵袭力；(3)伤寒沙门菌5'-UTR-malS为启始码上游448nt部分，对数早期表达较高，能影响对侧靶基因bax的mRNA的稳定性，下调Bax蛋白表达，而Bax有负向调控细菌动力和侵袭力的作用，说明5'-UTR-malS可通过下调Bax表达而促进细菌的动力和侵袭力。研究证实了伤寒沙门菌能通过三种不同的长链非编码RNA在不同的时期表达调节，发挥了生长早期有利于细菌的毒力，生长中后期有利于细菌应对环境酸、氧应激的作用，与Sigma因子RpoE、RpoS、RpoH以及调节因子OyxR等调节因子等共同组成影响细菌包括动力和侵袭力在内的毒力调节网络。研究发现获得了对伤寒沙门菌毒力调控与环境应激、重要调节因子作用关系的新认识，为今后设计控制细菌毒力及感染的方案与策略奠定了良好基础。