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Identification of a novel LysR-type transcriptional regulator in Staphylococcus aureus that is crucial for secondary tissue colonization during metastatic bloodstream infection

ABSTRACT: Purpose: The goal of this study was to identify Staphylococcus aureus genes which are important during establishment of metastatic infections in bloodstream infections. Methods: Pooled mariner transposon mutant libraries were generated as previously described (PMID: 27185949). The libraries were sequenced on the Illumina HiSeq 2500 platform with the transposon-specific oligonucleotide primer Himar1-Seq. Illumina adapter sequences were removed via cutadapt version 1.2.1. The reads were filtered for size (>16 bp) and to contain the signature transposon inverted repeat. Reads were mapped to the Staphylococcus aureus 6850 genome (RefSeq accession NC_022222.1) by Bowtie2 v2.1.0. Identification of depleted and enriched mutants was performed via DESeq2 version 1.6.2. Results: S. aureus genes were identified which were upregulated or downregulated upon iduced expression of the LysR-type transcriptional regulator RSAU_000852 in Staphylococcus aureus 6850. Conclusions: Our study identifies a gene for a LysR-type transcription regulator, which is involved in metabolic adaptation of the pathogen during colonization of secondary infection sites in a bloodstream infection model.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE139513 | GEO | 2020/09/08

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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Identification of a novel LysR-type transcriptional regulator in Staphylococcus aureus that is crucial for secondary tissue colonization during metastatic bloodstream infection

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