Name: Staphylococcus aureus ranalexin iTRAQ experiment (10 salt cuts)
Keywords: Not available

Dataset Information

Staphylococcus aureus ranalexin iTRAQ experiment (10 salt cuts)

ABSTRACT: Not available

INSTRUMENT(S): QSTAR XL, instrument model

ORGANISM(S): Staphylococcus Aureus (strain Mrsa252)

SUBMITTER: Catherine Botting

PROVIDER: PRD000312 | Pride | 2011-09-02

REPOSITORIES: Pride

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Publications

Global network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus.

Overton Ian M IM Graham Shirley S Gould Katherine A KA Hinds Jason J Botting Catherine H CH Shirran Sally S Barton Geoffrey J GJ Coote Peter J PJ

BMC systems biology 20110512

<h4>Background</h4>Staphylococcus aureus is a major human pathogen and strains resistant to existing treatments continue to emerge. Development of novel treatments is therefore important. Antimicrobial peptides represent a source of potential novel antibiotics to combat resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA). A promising antimicrobial peptide is ranalexin, which has potent activity against Gram-positive bacteria, and particularly S. aureus. Understanding mo ...[more]

PMID: 21569391

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Project description:The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics, and by its fast evolution into new lineages that rapidly spread within the healthy human population. This calls for the development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available as yet, the present studies were aimed at identifying new leads for their development. For this purpose, we thoroughly profiled the cell surface-exposed staphylococcal proteome by combining two surface shaving approaches. In parallel, non-covalently cell wall-bound proteins were extracted with KSCN and analyzed by gel-free proteomics, and also the exoproteome was analyzed through gel-free proteomics. Lastly, we screened a selection of the identified cell wall-attached proteins for binding of immunoglobulin G from patients that have been challenged with different types of S. aureus over extended periods of time due to chronic wound colonization. The combined results of these analyses highlight particular cell surface-exposed S. aureus proteins with highly immunogenic epitopes as potentially powerful targets for the development of protective anti-staphylococcal immunization strategies. Bioinformatics pipeline: Reduction and alkylation, desalting of the samples, mass spectrometric (MS) analysis and database searches were performed as previously described (PMID: 20662103). The strain-specific uniprot databases were used for the /S. aureus/ strains Newman and USA300, respectively including concatenated reversed databases with 5250 and 5298 entries. Validation of MS/MS-based peptide and protein identifications was performed with Scaffold (version Scaffold_2_04_00, Proteome Software Inc., Portland, OR). Peptide identifications were accepted if they exceeded the specific database search engine thresholds. Sequest identifications required at least deltaCn scores of greater than 0.10 and XCorr scores of greater than 1.9, 2.2, 3.8 and 3.8 for singly, doubly, triply and quadruply charged peptides, respectively. All experiments were conducted in independent triplicates. Peptides were only accepted as identified if they were detected in at least two out of the three replicates per sample set. With these filter parameters no false positive hits were obtained.

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 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.

Dataset Information

Staphylococcus aureus ranalexin iTRAQ experiment (10 salt cuts)

Publications

Global network analysis of drug tolerance, mode of action and virulence in methicillin-resistant S. aureus.

Similar Datasets

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