Proteomic profiling of cell envelope-associated proteins from Staphylococcus aureus.
The emergence of highly virulent community acquired Staphylococcus aureus and continued progression of resistance to multiple antimicrobials, including methicillin and vancomycin, marks the reemergence 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 2-DE or chromatographic separation of proteins with 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, four of which are uncharacterized to date. One of the two most abundant cell envelope proteins, which showed remarkably high variations in MW and pI in the 2-DE gel display, was the S. aureus surface protein G. 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. We describe a novel semiquantitative method to determine abundance factors of such proteins in 2-DE gels of cell envelope fractions relative to whole cell lysates and discuss these data in the context of true cell envelope localization versus experimentally caused cell lysis.
Gatlin
Christine L
CL
The Institute for Genomic Research, Rockville, MD, USA. clgatl@wm.edu
Pieper
Rembert
R
Huang
Shih-Ting
ST
Mongodin
Emmanuel
E
Gebregeorgis
Elizabeth
E
Parmar
Prashanth P
PP
Clark
David J
DJ
Alami
Hamid
H
Papazisi
Leka
L
Fleischmann
Robert D
RD
Gill
Steven R
SR
Peterson
Scott N
SN
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013211:Staphylococcus aureus / Q000737:chemistry* / Q000166:cytology; D013347:Subcellular Fractions / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D010455:Peptides; D020543:Proteome