Expression profile analyses and target identification of the small RNA MarS in Streptococcus pyogenes: a transcriptome based study

化脓性链球菌是一种具有极其多样化临床表现的高全球疾病负担。化脓性链球菌感染的成功取决于大量严格控制的毒力因子。小的非编码rna （sRNAs）属于负责细菌基因调控的调控分子。在致病性生物中，sRNAs经常参与控制毒力基因的表达。最近，我们在化脓性葡萄球菌中发现了调节中心转录激活因子基因mga表达的反式作用sRNA MarS。在没有MarS的情况下，化脓性葡萄球菌粘附人角质形成细胞或在人血液中存活的能力减弱。在小鼠感染模型中，粘附性促进毒力因子的下调导致marS缺失突变体的传播增加。典型地，反式作用的srna靶向不止一种特定的mRNA。在这个项目中，我们的目标是扩大MarS调控和分子机制的表征。为了实现这些目标，我们将分两个步骤进行。首先，我们将研究在什么条件下marS在化脓性链球菌中表达。该方法将使用三种不同的血清型，代表三种不同的链球菌疾病临床表现：M1T1菌株MGAS5005， M18菌株MGAS8232和M49菌株591。细菌将在不同的生长阶段和暴露于不同的感染相关的应激条件下取样。RNA-seq将用于研究这三种菌株在不同条件下的转录组。因此，可以监测3株菌株中marS的表达谱。同时，通过在不同条件下生长后混合RNA制剂从每个菌株中获得的RNA池将用于分析和比较整体转录组组织。这些数据将提供有关转录起始位点、操纵子结构、反义转录的信息，并将导致鉴定新的sRNA候选基因。在第二步，我们将通过比较野生型菌株与其等基因的火星缺失菌株来确定假定的火星目标。细菌将在确定的促进marS表达的条件下培养。RNAseq将用于检测差异表达基因，这些基因代表假定的靶标。计算机预测程序（IntaRNA, CopraRNA）将用于支持候选靶mrna与火星直接相互作用的研究。预测结果有助于设计用于sRNA-mRNA凝胶转移试验的RNA探针。假定的MarS靶点的生理相关性将通过体外毒力测定来研究。

Streptococcus pyogenes is responsible for a high global disease burden with extremely diverse clinical manifestations. The success of S. pyogenes infections depends on an immense repertoire of strictly regulated virulence factors. Small non-coding RNAs (sRNAs) belong to the regulatory molecules responsible for bacterial gene regulation. In pathogenic organisms sRNAs are frequently involved in the control of virulence gene expression. Recently, we identified the trans-acting sRNA MarS, which modulates the expression of the central transcriptional activator gene mga, in S. pyogenes. In the absence of MarS, the ability of S. pyogenes to adhere to human keratinocytes or to survive in human blood was diminished. Down-regulation of adherence promoting virulence factors led to an increased dissemination of the marS deletion mutant in a murine infection model. Typically, trans-acting sRNAs target more than one specific mRNA. In this project we aim at the expansion of the MarS regulon and at the characterization of the molecular mechanism. To meet these objectives we will proceed in two steps. First, we will investigate under which conditions marS is expressed in S. pyogenes. Three different serotypes, representing three different clinical manifestations of streptococcal disease, will be used for this approach: M1T1 strain MGAS5005, M18 strain MGAS8232, and M49 strain 591. Bacteria will be sampled during different growth phases and following exposure to diverse infection-relevant stress conditions. RNA-seq will be performed to study the transcriptome of the three strains during the various conditions. Thereby, the expression profile of marS can be monitored in the three strains. In parallel, a pool of RNA, obtained from each strain by mixing RNA preparations following growth under the different conditions, will be used for the analyses and comparison of overall transcriptome organization. These data will provide information about transcriptional start sites, operon structures, antisense transcription, and will lead to the identification of novel sRNA gene candidates. In the second step, we will identify putative MarS targets by comparing wildtype strains to their isogenic marS deletion strains. Bacteria will be cultivated under conditions which were determined to promote marS expression. RNAseq will be performed to detect differentially expressed genes, which represent putative targets. In silico prediction programs (IntaRNA, CopraRNA) will be used to support investigation of direct interaction of the candidate target mRNAs and MarS. Prediction results facilitate the design of RNA probes for sRNA-mRNA gel-shift assays. Physiological relevance of putative MarS targets will be studied by in vitro virulence assays.