Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of Newman delta2027

ABSTRACT: Intracellular small-molecule signaling has drawn increasing attention due to its importance in bacterial physiology and host-pathogen interactions. Here, we show that a new quinone-sensing transcriptional regulator, QsbR (quinone-stress sensing and bleomycin resistance regulator), is important for bacterial stress response and virulence in S. aureus. The mutant strain of qsbR exhibits dramatically reduced staphylococcal virulence in a murine abscess model of infection. Moreover, the qsbR mutant shows an accumulation of riboflavin and an increased resistance to quinone stress and bleomycin compared to the wild-type strain Newman. Microarray analysis confirmed that QsbR impacts the expression of genes that are involved in quinone stress response, bleomycin resistance, riboflavin biosynthesis, and pathogenesis. DNase-I footprinting and gel shift assays suggest that QsbR not only directly regulates transcription of genes involved in quinone detoxification and bleomycin resistance, but is also subject to autoregulation by binding to the palindromic consensus sequence GTATAN(5)TATAC in their promoter regions. This binding could be disrupted by quinone treatment in vitro. Mass spectrometric analysis showed that wild-type QsbR, but not QsbRC5S with Cys5 mutated to Ser, could be alkylated at Cys5 by quinone. Further mutagenesis studies revealed that Cys5 plays a key role in quinone sensing as substitution of Cys5 with Ser renders the bacterium more sensitive to quinone stress. Our results demonstrate that QsbR is a quinone-sensing regulator that dramatically impacts staphylococcal virulence and stress response. Sample preparation for transcriptional analysis. For each strain, 2 clones were inoculated in TSB and collected from mid-log or stationary phase. Appropriate volumes from each of the 2 cultures per strain were pooled and added to 2 volumes of RNAprotect reagent (Qiagen). Cultures were centrifuged and cell pellets were stored at 4 ÂºC until RNA extraction. During the experiment, OD600 and viable cfu per ml were monitored for each of the cultures. This procedure was repeated 2 independent times to generate 2 samples at each time point for each strain.

ORGANISM(S): Staphylococcus aureus subsp. aureus str. Newman

SUBMITTER: John Braisted

PROVIDER: E-GEOD-35409 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 NM3, 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. Staphylococcus aureus subsp.aureus strain Newman (reference) and Staphylococcus aureus subsp.aureus strain Newman yhcR knockout(query) were grown in TSA broth.Samples were grown under aerobic and anaerobic conditions and RNA samples harvested at mid log, stationary, and log phases.Samples were hybridized on aminosilane coated slides with 70-mer oligos.

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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Transcriptome analysis of Newman delta2027

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