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Staphylococcus aureus Resistance to Vancomycin
Comparative Proteomic Analysis of Staphylococcus aureus Strains with Differences in Resistance to the Cell Wall-Targeting Antibiotic Vancomycin
Rembert Pieper, Christine L. Gatlin-Bunai, Emmanuel F. Mongodin, Prashanth P. Parmar, Shih-Ting Huang, David J. Clark, 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
Three isogenic strains derived from a clinical vancomcyin-intermediate Staphylococcus aureus (VISA) isolate were examined by comparative protein abundance analysis. Subcellular fractionation was followed by protein separation in 2-DE gels and spot identification by MALDI-TOFTOF-MS and LC-MS/MS. Sixty-five significant protein abundance changes were determined. Numerous enzymes participating in the purine biosynthesis pathway were dramatically increased in abundance in strain VP32, which featured the highest minimal inhibitory concentration for vancomycin, compared to strains P100 and HIP5827. Peptidoglycan hydrolase LytM (LytM) and the SceD protein, a putative transglycoslylase, were increased in abundance in the cell envelope fraction of strain VP32, whereas the enzyme D-Ala--D-Ala ligase was decreased in its cytosol fraction. Furthermore, penicillin-binding protein 2 (PBP2) had substantially higher activity in strain VP32 compared to that in strain HIP5827. LytM, PBP2 and D-Ala--D-Ala ligase catalyze reactions in the biosynthesis or the metabolism of cell wall peptidoglycan. It is plausible that expression and activity changes of these enzymes in strain VP32 are responsible for an altered cell wall turnover rate, which has been observed, and an altered peptidoglycan structure, which has yet to be elucidated for this highly vancomycin-resistant strain.
Description of Figures and Tables
A 2-DE gel-based approach was used for differential protein abundance profiling of three S. aureus strains derived from a virulent clinical isolate (HIP5827). HIP5827 was described as a clinical vancomycin intermediate resistant S. aureus (VISA) isolate with a minimal inhibitory concentration (MIC) of 8 µg/mL vancomcyin. In addition to HIP5827, two isogenic strains were obtained by passage in the absence of vancomcyin (strain P100, MIC of 2 µg/mL vancomcyin) and with increased concentrations of vancomcyin (strain VP32, 32 µg/mL vancomcyin). The objective was profile the bacterial proteome of the VISA strains separately in a cytoplasmic and a cell envelope fraction. Enrichment of proteins from the cell envelope was of particular interest due to cell wall thickness and structural changes known to occur in VISA strains compared to antibiotic-sensitive strains. In a previous study, it had been determined that P100, HIP5827 and VP32 (in this order) featured increasing cell wall thickness and reduced cross-linking. Our proteomic dataset was compared to that of a transcriptional profiling analysis via DNA microarrays.
Highly significant changes were not observed comparing strains P100 (low MIC) and HIP5827 (intermediate MIC). In Figures 1 and 2, significant protein spot changes (>1.5 fold ratio) comparing strain P100 and VP32 cytoplasmic fractions are displayed. In Figure 3, significant protein spot changes (>1.5 fold ratio) comparing strain P100, HIP5827 and VP32 cell envelope fractions are displayed in montage view. Descriptions related to the isolation of the cell envelope fraction, are provided in the VISA cell envelope profiling study (see website). The spots denoted in the three Figures are listed in numerical order in the Table. The Table not only provides the accession numbers and names for differentially abundant proteins, but also a variety of data derived from 2-DE gels, differential gel image analysis, mass spectrometry and transcript abundance data. The most dramatic abundance changes observed in the differential protein spot analysis were attributed to purine biosynthesis pathway enzymes (spots 1-19), which were up-regulated in VP32, the most vancomcyin-resistant strain, and proteins expressed from a S. aureus mobile genetic element which harbors known or putative antibiotic resistance genes and virulence factors (spots 43-47), which were down-regulated in VP32. Currently, the implications of these protein abundance changes with respect to vancomcyin resistance are not known. A known peptidoglycan hydrolase, LytM, was found to be increased in strain VP32, but only for a spot corresponding to the Mr and pI of the LytM proenzyme. Via zymography, we were unable to determine whether its enzymatic activity was increased in strain VP32. Its activity change could explain the differences in cell wall-crosslinking comparing the three VISA strains. Zymography did not reveal major changes in other cell wall-hydrolytic activities, e.g. for the prominent bifunctional autolysin Atl. A zymographic analysis is displayed in Figure 5. A indicated, a few major hydrolytic bands were cut from zymograms and proteins identified by LC-MS/MS. Penicillin-binding proteins (PBPs) were not reliably profiled in 2-DE gel images, likely due to hydrophobic regions and basic pIs. In a substrate-labeling and SDS-PAGE analysis experiment, PBPs were detected as fluorescence-labeled bands. Increased activities of the enzyme PBP-2 in strain VP32 were verified by LC-MS/MS. Cell wall assembly and crosslinking functions have been previously attributed to this enzyme, particularly in highly vancomcyin-resistant S. aureus (VRSA) strains. As shown in Figure 4, the increased PBP activity in strain VP32 was detected in both cell envelope and cell membrane fractions.
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