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

化脓性链球菌是全球疾病负担最大的病原菌，其临床表现极为多样。化脓性链球菌感染的成功依赖于大量严格调控的毒力因子。小分子非编码RNA(SRNAs)是负责细菌基因调控的调节分子。在病原生物中，sRNAs经常参与毒力基因表达的调控。最近，我们在化脓性链球菌中发现了调控中枢转录激活基因MGA表达的反式作用基因SRNA MARS。在没有MARS的情况下，化脓性链球菌附着在人类角质形成细胞上或在人类血液中生存的能力减弱。在小鼠感染模型中，下调依从性促进毒力因子导致MARS缺失突变体的传播增加。通常，反式作用的sRNA针对不止一个特定的mRNAs。在这个项目中，我们的目标是MARS调节子的扩展和分子机制的表征。为了实现这些目标，我们将分两个步骤进行。首先，我们将研究在哪些条件下MARS在化脓性链球菌中表达。这一方法将使用三种不同的血清型，代表链球菌疾病的三种不同临床表现：M1T1株MGAS5005，M18株MGAS8232和M49株591。在不同的生长阶段和暴露在与感染相关的各种压力条件下后，将对细菌进行采样。RNA-SEQ将用于研究这三个菌株在不同条件下的转录组。因此，可以监测MARS在三个菌株中的表达谱。同时，通过混合在不同条件下生长的RNA制剂从每个菌株获得的RNA池将用于分析和比较整个转录组组织。这些数据将提供有关转录起始点、操纵子结构、反义转录的信息，并将导致新的sRNA候选基因的鉴定。在第二步中，我们将通过将野生型菌株与其同源MARS缺失菌株进行比较来确定可能的MARS靶标。细菌将在被确定为促进MARS表达的条件下培养。RNAseq将被用来检测差异表达的基因，这些基因代表了可能的靶标。在计算机预测程序(IntaRNA、CopraRNA)中，将用于支持候选目标mRNAs与MARS的直接相互作用的研究。预测结果有助于设计用于sRNA-mRNA凝胶位移分析的RNA探针。可能的MARS靶标的生理相关性将通过体外毒力分析进行研究。

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.