The Small Noncoding RNA, RsaC, Is Essential for Staphylococcus aureus Virulence.

Abstract

Background: Bacterial small noncoding RNAs (sRNAs) play critical roles in virulence, stress adaptation, and host-pathogen interactions. Transcriptomic analyses during infection can help reveal pathogen-derived sRNAs required for pathogenesis, providing valuable insights for the development of novel therapeutic strategies. However, the low abundance of pathogen biomass within the host tissues poses a significant challenge for such analyses.

Methods: We employed 2-step cell disruption to enrich Staphylococcus aureus cells from infected mouse organs and conducted RNA sequencing (RNA-seq) analysis to examine staphylococcal sRNAs expressed during infection. qRT-PCR was used to confirm the gene expression. A knockout mutant of highly expressed sRNA, RsaC, was generated, and RNA-seq under in vivo as well as in vitro aerobic and anaerobic conditions were compared between the wild-type and ΔrsaC strains. Virulence of S. aureus was assessed using both mouse and silkworm survival assays.

Results: We identified RsaC as one of the most highly expressed sRNAs in mouse organs with consistent increment over time postinfection. Through gene disruption and complementation, we demonstrated that RsaC is an independent virulence determinant required for full pathogenicity of S. aureus in a murine infection model. In addition, RsaC influenced gene expression in response to oxygen availability and host-associated stress. Further analysis revealed that mutation of 2 genes downregulated in ΔrsaC in vivo, NWMN_RS03420 (sodium: proton antiporter) and NWMN_RS12015 (hypothetical protein), reduced S. aureus virulence in a silkworm model.

Conclusions: These findings identify RsaC as a novel independent virulence determinant that supports S. aureus adaptation within the host.