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

化脓性链球菌中小 RNA MarS 的表达谱分析和靶点鉴定：基于转录组的研究

• 批准号: 420051101
• 负责人: Privatdozentin Dr. Nadja Patenge, Ph.D.
• 金额: --
• 依托单位: Institut für Medizinische Mikrobiologie, Virologie und Hygiene
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/420051101?language=en
• 关键词: Expression; profile; analyses; target; identification

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.

化脓性链球菌造成全球高疾病负担，临床表现极其多样化。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。小非编码RNA（sRNA）属于负责细菌基因调控的调控分子。在病原生物体中，sRNA 经常参与毒力基因表达的控制。最近，我们在化脓性链球菌中鉴定出了反式作用 sRNA MarS，它调节中央转录激活基因 mga 的表达。在没有 MarS 的情况下，化脓性链球菌粘附于人角质形成细胞或在人血液中存活的能力就会减弱。在小鼠感染模型中，粘附促进毒力因子的下调导致 marS 缺失突变体的传播增​​加。通常，反式作用 sRNA 靶向不止一种特定 mRNA。在这个项目中，我们的目标是扩展 MarS 调节子并表征分子机制。为了实现这些目标，我们将分两步进行。首先，我们将研究 marS 在什么条件下在化脓性链球菌中表达。该方法将使用代表链球菌疾病三种不同临床表现的三种不同血清型：M1T1 菌株 MGAS5005、M18 菌株 MGAS8232 和 M49 菌株 591。将在不同的生长阶段和暴露于不同的感染相关应激条件后对细菌进行取样。将进行 RNA-seq 以研究三种菌株在不同条件下的转录组。因此，可以监测三个菌株中 marS 的表达谱。与此同时，通过混合在不同条件下生长后的RNA制剂从每个菌株获得的RNA池将用于整体转录组组织的分析和比较。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，并将导致新型 sRNA 基因候选物的鉴定。在第二步中，我们将通过将野生型菌株与其等基因 marS 缺失菌株进行比较来确定假定的 MarS 靶点。将在确定促进marS表达的条件下培养细菌。将进行 RNAseq 来检测代表假定目标的差异表达基因。计算机预测程序（IntaRNA、CopraRNA）将用于支持候选目标 mRNA 和 MarS 直接相互作用的研究。预测结果有助于设计用于 sRNA-mRNA 凝胶迁移测定的 RNA 探针。将通过体外毒力测定来研究假定的 MarS 靶标的生理相关性。