Expression profile analyses and target identification of the small RNA MarS in Streptococcus pyogenes: a transcriptome based study
Final Report Abstract
Streptococcus pyogenes is responsible for a high global disease burden with a wide range of clinical manifestations. The success of S. pyogenes infections depends on an extensive repertoire of tightly regulated virulence factors. Small non-coding RNAs (sRNAs) are among the regulatory molecules involved in the control of virulence factor gene expression in pathogenic bacteria. In preliminary work for this proposal, we identified the trans-encoded sRNA MarS, which modulates expression of the gene encoding the key transcriptional activator Mga in S. pyogenes, influences S. pyogenes adherence to human keratinocytes, survival in human whole blood, and dissemination in the murine infection model. In this project, marS expression was investigated to identify additional target mRNAs. To this end, conditions under which marS was expressed in S. pyogenes were determined. Three different serotypes representing three different clinical manifestations of streptococcal disease were used for this approach: M3 (MGAS315), M18 (MGAS8232), and M49 (591). Bacteria were cultured under different conditions and harvested at different growth stages and after exposure to infection-related stress. The serotype-specific expression profiles of marS under the different conditions were determined by reverse transcription followed by quantitative PCR. While marS expression remained constant across growth phases, it was reduced in late stationary phase, under glucose deficiency, pH stress, and oxidative stress. Increased marS expression was observed during temperature stress. In addition, one RNA pool per strain from samples of all conditions was analyzed using RNAseq to analyze and compare transcriptome organization. Genome-wide transcription starts, operon structures, antisense transcription, phage regions, and sRNA genes were identified. Information from the transcriptome data was used to produce isogenic marS deletion mutants and complementation strains. For complementation, marS was integrated into transcriptionally silent regions of the genome under the control of its own promoter to avoid overproduction of the small RNA, which had led to high data variability in the past. Deletion mutants are now available for all three strains, as well as complementation strains that show wild-type marS expression levels and will be used to identify MarS target mRNAs and applied for functional studies. Bacteria were cultured under conditions that influence marS expression and RNAseq was used to detect differentially expressed genes that are potential targets. RNAseq data analyses and verification of the physiological relevance of putative targets are currently ongoing. In summary, by providing the global RNA landscape and analyzing marS expression profiles in three relevant S. pyogenes serotypes, this project substantiated the cross-serotype role of marS. Furthermore, by providing improved deletion and complementation constructs, the stage was set for detailed analysis of the marS regulon.
Publications
-
Whole-Genome Sequence of Streptococcus pyogenes Strain 591, Belonging to the Genotype emm 49. Microbiology Resource Announcements, 10(43).
Patenge, Nadja; Rückert, Christian; Bull, Jana; Strey, Kevin; Kalinowski, Joern & Kreikemeyer, Bernd
