Transcriptomic exploration of SSR42 and its impact on Staphylococcus aureus virulence

Perhaps the most pivotal feature contributing to Staphylococcus aureus pathogenicity is the production of a wealth of virulence factors such as secreted toxins, adhesive proteins, and factors that aid in host immune evasion. The abundance and activity of these factors is tightly regulated by a multitude of effectors, including two-component systems, alternative sigma factors, DNA binding transcription factors, and regulatory RNAs. SSR42 is a ~1,200-nt long regulatory RNA that has been previously shown to be highly stable and abundant in stationary phase growth. To explore the role of SSR42 within the S. aureus cell, we created a full deletion mutant for this gene and performed RNA sequencing analysis after 15h of growth. Following standardized bacterial growth and sample processing, total bacterial RNA was isolated utilizing the Qiagen RNeasy mini kit and contaminating DNA was removed by treatment with Ambion DNA-free kit. Total RNA was enriched for mRNA using MicrobExpress kit and Illumina RiboZero rRNA removal kit for Gram-positive bacteria. Library construction was performed using the TruSeq Stranded mRNA Kit from Illumina, and RNA was sequenced using an Illumina Nextseq 550. When compared to wild-type, the SSR42 mutant exhibited profound changes in its transcriptomic landscape, suggesting that SSR42 has myriad regulatory targets across niches of function. Overall design: S. aureus RNA was extracted to compare global gene expression of two samples: HOU wildtype and HOU SSR42 deletion mutant. HOU wildtype serves as the parental control strain. Each sample was generated by pooling three independent biological replicate RNA preps.