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.

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.

Project description:Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). ?B is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that ?B is required for polysaccharide-independent biofilms, controversy persists over the role of ?B in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus ?B-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the ?B mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active ?B, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.IMPORTANCE Due to its multifaceted lifestyle, Staphylococcus aureus needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named ?B (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of ?B in the regulation of the biofilm lifestyle in S. aureus Our study demonstrated that ?B is not necessary for exopolysaccharide-dependent biofilms and, even more, that S. aureus produces stronger biofilms in the absence of ?B The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.

Dataset Information

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

Publications

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

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