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

• Applicant: Privatdozentin Nadja Patenge, Ph.D.
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: from 2019 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 420051101

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.

• DFG Programme: Research Grants