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Transcriptomic exploration of SSR42 and its impact on Staphylococcus aureus virulence

ABSTRACT: 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.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE237701 | GEO | 2023/07/25

REPOSITORIES: GEO

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Project description:Staphylococcus aureus is a Gram-positive human pathogen causing a variety of human diseases in both hospital and community settings. This bacterium is so closely associated with prophages that it is rare to find S. aureus isolates without prophages. Two phages are known to be important for staphylococcal virulence: the beta-hemolysin (hlb) converting phage and the Panton-Valentine Leukocidin (PVL) converting phage. The hlb-converting phage is found in more than 90% of clinical isolates of S. aureus. This phage produces exotoxins and immune modulatory molecules, which inhibit human innate immune responses. The PVL-converting phage produces the two-component exotoxin PVL, which can kill human leucocytes. This phage is wide-spread among community-associated methicillin resistant S. aureus (CA-MRSA). It also shows strong association with soft tissue infections and necrotizing pneumonia. Several lines of evidence suggest that staphylococcal prophages increase bacterial virulence not only by providing virulence factors but also by altering bacterial gene expression: 1) Transposon insertion into prophage regulatory genes, but not into the genes of virulence factors, reduced S. aureus killing of Caenorhabditis elegans.; 2) Although the toxins and immune modulatory molecules encoded by the hlb- converting phages do not function in the murine system, deletion of ϕNM3, the hlb-converting phage in S. aureus Newman, reduced staphylococcal virulence in the murine abscess formation model. 3) In a preliminary microarray experiment, prophages in S. aureus Newman altered the expression of more than 300 genes. In this research proposal, using microarray and high-throughput quantitative RT-PCR (qRT-PCR) technologies, we will identify the effects of the two important staphylococcal phages on the gene expression of S. aureus in both in vitro and in vivo conditions. This project is intended to be completed within one year. All the data – microarray, qRT-PCR and all the primer sequences- will be made available to public 6 month after completion. Data from this project will help us to understand the role of prophages in the S. aureus pathogenesis and can lead to development of a strategy to interfere with the pathogenesis process. Following strains were grown in TSA broth: Staphylococcus aureus USA300 (reference) Staphylococcus aureus USA300 with deletion of ϕSa2usa (Query) Staphylococcus aureus USA300 with deletion of ϕSa3usa (Query) Staphylococcus aureus USA300 Prophage-free mutant (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa2mw (Query) Staphylococcus aureus USA300 Prophage-free mutant lysogenized with ϕSa3usa (Query) strain: Staphylococcus aureus USA300 Prophage-free mutant lysogenized with both ϕSa2mw and ϕSa3usa (Query) RNA samples were harvested at early log, midlog and stationary phase.Samples were hybridized on aminosilane coated slides with 70-mer oligos.

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Transcriptomic exploration of SSR42 and its impact on Staphylococcus aureus virulence

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