氨苄西林压力下ncRNAs参与伤寒沙门菌RpoE负向调控ramA机制研究

RpoE, an important sigma factor, can initiate some important gene expressions under environment stresses, and also negatively regulate some target gene expressions through promoting non-coding RNA (ncRNA). We found that the rpoE mutant of Salmonella enterica, serovar Typhi (S. Typhi) had elevated resistance to several antimicrobial agents including ampicillin when compared to the wild-type strain, mainly through the increasing expression of drug efflux pump regulator RamA. Based on the reports from the others and our group, it is suggested that dysfunction of RpoE in S. Typhi could reduce the expression of some ncRNAs, which in turn increasing the expression of ramA. In this proposal, the strand-specific RNA High-throughput sequencing will be employed to analyze the transcriptome of wild-type S. Typhi under ampicillin pressure. In combination to reference whole genome sequencing analysis, we will identify the ncRNAs expression profiles of S. Typhi under ampicillin pressure. Oligo microarray and real-time RT-PCR will then be used to systematically analyze the expression of ncRNAs in rpoE defective mutant under ampicillin pressure. Furthermore, we will examine if RpoE could negatively regulate ramA through ncRNAs under ampicillin pressure using gene knockout and complementation assaies. The results of this study will deep our understanding in the genetic basis of antibiotic resistance in Salmonella enterica serovar Typhi.

RpoE是肠道菌在环境应激下启动基因表达的重要Sigma因子，也可通过启动非编码RNA(ncRNA)表达进而负向调控靶基因。我们发现，伤寒沙门菌rpoE缺陷株相比野生株，通过上调药物外排泵调节子RamA表达，从而表现出对包括氨苄西林在内的多种抗生素耐受水平上升；结合文献及课题组前期研究结果，推测可能rpoE缺失导致某些ncRNAs的表达降低，进而实现ramA基因的高表达。本项目拟利用链特异性高通量测序技术，分析伤寒沙门菌野生株在氨苄西林压力下的转录组，参比同一菌株全基因组精细结构，确定其在氨苄西林压力下的ncRNAs基因组；进而应用Oligo芯片和qRT-PCR技术，系统分析rpoE缺陷变异株在氨苄西林压力下ncRNAs的表达变化；再利用基因敲除与回补等技术，进一步验证氨苄西林压力下伤寒沙门菌ncRNAs参与RpoE对ramA基因表达的负向调节作用。本研究将促进对伤寒沙门菌耐药机制的认识。

RpoE是肠道菌在环境应激下启动基因表达的重要Sigma因子，也可通过启动非编码RNA(ncRNA)表达进而负向调控靶基因。前期研究显示伤寒沙门菌rpoE缺陷株相比野生株对包括氨苄西林在内的多种抗生素耐受水平上升，可能与ncRNA表达有关。本研究通过基因敲除、基因回补、高表达等技术，明确了RpoE通过负向调控药物外排泵调节子RamA，正向调控外膜蛋白OmpF、OmpC的表达参与伤寒沙门菌耐受氨苄西林。证实了t2288（RamR）不是ramA的转录抑制因子，不影响ramA对氨苄西林抗生素的耐受，不参与ramA相关的耐药机制调控。利用链特异性高通量测序技术，分析伤寒沙门菌野生株在氨苄西林压力下的转录组，参比同一菌株全基因组精细结构，确定其在氨苄西林压力下的ncRNAs基因组；并进一步利用测序及qRT-PCR技术，系统分析了rpoE缺陷变异株在氨苄西林压力下ncRNAs的表达变化，筛选到受RpoE 调控的ncRNA36个，包括scaffold46_minus_1、scaffold8_minus_1、scaffold88_minus_1、 scaffold81_plus_1等。进一步利用qRT-PCR、Northern-blot等技术证实了RpoE通过对ncRNA scaffold46_minus_1的影响实现对ramA的负向调控。本研究对伤寒沙门菌在氨苄西林压力下，关键调节因子发挥作用的机制进行了深入研究，明确了氨苄西林压力下伤寒沙门菌ncRNA参与RpoE对ramA基因表达的负向调节作用。研究结果促进了对伤寒沙门菌耐药机制的认识，也为研究其它肠道菌的耐药机制提供了理论基础，对临床预防和治疗耐药菌株具有重要的参考价值。