Three New Bioinformatics Tools Available
The NIAID sponsored Pathogen Functional Genomics Resource Center (PFGRC) at the J. Craig Venter Institute is pleased to announce the release of three new, free open-source software tools: Magnolia, Ginkgo and APEX. Magnolia is a microarray data management and export system for researchers who use PFGRC microarrays. The software greatly simplifies the tasks of organizing experimental data and submitting it to a public data repository. Ginkgo is a Comparative Genomic Hybridization (CGH) and expression microarray data analysis package. Several normalization, data filtering and imputation, and replicate microarray functions are implemented in an intuitive graphical framework. The APEX tool is an implementation of the Absolute Protein Expression quantitation technique. It can compute protein abundance values for LC-MS/MS proteomics datasets, quantifying hundreds or thousands of proteins. Links to additional information on each of these new software tools is available from the PFGRC's bioinformatics page.
Microarray Suggestion Criteria
The National Institute of Allergy and Infectious Diseases (NIAID) supported Pathogen Functional Genomics Resource Center (PFGRC) designs, constructs, and distributes glass slide DNA microarrays for pathogens and biodefense related organisms (Select A-C agents). Currently, the PFGRC supports DNA microarrays for the 38 organisms listed here. In continuing its efforts to provide the infectious disease and biodefense communities with the microarray resources most relevant to their research efforts, the PFGRC is soliciting input for selection of its next set of reference/species microarrays. The criteria for organism selection may be found here.
Staphylococcus aureus Cell Envelope-Associated Proteins
Proteomic Profiling of Cell Envelope-Associated Proteins from Staphylococcus aureus
Christine L. Gatlin, Rembert Pieper, Shih-Ting Huang, Emmanuel Mongodin, Elizabeth Gebregeorgis, Prashanth P. Parmar, David J. Clark, Hamid Alami, Leka Papazisi, Robert D. Fleischmann, Steven R. Gill, Scott N. Peterson
The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, U.S.A. Correspondence:
Rembert Pieper, Pathogen Functional Genomics Resource Center, The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850, U.S.A., E-mail: rpieper@tigr.org
Abstract
The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the re-emergence of S. aureus as a serious health care threat. Investigation of proteins localized to the cell surface could help to elucidate mechanisms of virulence and antibiotic resistance in S. aureus. In this study, proteomic profiling methods were developed to solubilize, display and evaluate abundance levels of proteins present in the supernatants of the lysostaphin-digested cell envelope from cultured vancomycin-intermediate S. aureus (VISA) cells. Combining approaches of two-dimensional gel electrophoresis (2-DE) or chromatographic separation of proteins with mass spectrometry (MS) analyses resulted in the identification of 144 proteins of particular interest. Of these proteins, 48 contained predicted cell wall localization or export signal motifs, including 14 with distinct covalent peptidoglycan-anchor sites, 4 of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in Mr and pI in the 2-DE gel display, was the S. aureus surface protein G (SasG). The display of numerous secreted proteins that are not covalently cell wall-anchored, suggests that, in the exponential growth phase, secreted proteins can be retained physiologically in the cell envelope and may interact with cell wall-anchored proteins and carbohydrate structures in a manner yet to be determined. The remaining 96 proteins, devoid of recognizable motifs, were repeatedly profiled in the VISA cell envelope fractions.
Description of Figures and Tables
The 2-DE gels in Figures 2-4 display profiles of proteins isolated from a vancomycin intermediate resistant Staphylococcus aureus (VISA) strain. Three different strategies were applied to isolate the proteins, two of them (neCWS and CWDS samples) targeted cell envelope-associated proteins. The CWDS sample was recovered from experiments in which the cell wall was digested with the enzymes lysostaphin and mutanolysin. Their enzymatic cleavage sites in the S. aureus cell wall are depicted in Figure 1. Lysostaphin specifically cleaves pentaglycine peptide bonds and releases a class of proteins which are covalently cell wall-anchored, LPXTG motif proteins. Those identified in our effort are primarily displayed in the 2-DE gel in Figure 3 and listed in one of the protein categories of Table 1. While many LPXTG proteins have not been thoroughly characterized, some are known to play roles in S. aureus virulence including biofilm formation, adhesion to host cells and extracellular matrix as well as immune evasion.
To assess which proteins are not covalently cell wall-linked, yet present in the cell envelope, a high salt extraction procedure was applied (neCWS sample, 2-DE gel in Figure 2). The comparison of spot patterns in Figures 2 and 3 shows dramatic differences indicating the prevalence of LPXTG motif proteins in the cell envelope. To confirm that the proteomic differences were not caused by cell lysis under the cell treatment conditions, a whole cell VISA lysate (WCL) was generated (displayed in the gel in Figure 4). Spot pattern comparisons (gel images in Figure 4 vs. Figures 2 and 3) and MS analysis verified that the cell envelope protein extraction / solubilization methods were selective. However, numerous proteins - typically in relatively small quantities - for which intracellular localizations have been predicted or experimentally demonstrated were also solubilized via salt and enzymatic extraction in the neCWS and CWDS samples, respectively. The proteins are denoted with ëBí spot numbers in Figures 2-4 and are listed in Table 2. In general, quantitative protein difference analyses did not allow us to reveal whether these intracellular proteins were truly present in the cell envelope of living cells, resulted from lysis of dead cells or from experimentally caused lysis. As shown in Table 1, secreted and membrane-associated proteins were also determined to be enriched in the cell envelope. Summarily, this dataset shows that proteomic analysis is useful to profile bacterial cell envelope proteins quantitatively. In particular, this study revealed the predominance of a cell surface protein called SasG in the VISA strain (gel in Figure 3). SasG shows extremely high post-translational modification levels. SasG had not been detected previously in high abundance in the cell envelope of an S. aureus strain. The question arises whether SasG is specific to VISA strains.
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S. aureus proteins not bioinformatically predicted to be secreted or present in the cell envelope |