Proteome Analysis of Membrane and Cell Wall Associated Proteins from Staphylococcus aureus

Abstract

Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.

The cell-envelope proteome of Bifidobacterium longum in an in vitro bile environment

“…In relation to this, with the aim of determining the protein profile of the B. longum envelope, and as a first approach to undertaking deeper functional studies, we analysed different subcellular fractions. The application of gel-based and gel-free technologies, combined with high-throughput techniques, allowed us to identify 218 proteins; about 70 % of them were predicted to be, or were previously described as being, in the cell envelope of Gram-positive bacteria (Antikainen et al, 2007;Candela et al, 2007;Eymann et al, 2004;Granato et al, 2004;Jang & Hanash 2003;Kelly et al, 2005b;Nandakumar et al, 2005;Rivera-Amill et al, 2001;Rodríguez-Ortega et al, 2006;Schaumburg et al, 2004;Severin et al, 2007;Silveira et al, 2004;Tjalsma et al, 2008;Wolff et al, 2007). Furthermore, 48 of them are predicted to be integral membrane proteins (contain hypothetical transmembrane segments) and 30 of them have different extracytoplasmic sorting signals.…”

Mapping the Pathways to StaphylococcalPathogenesis by Comparative Secretomics

“…Proteomics-based data sets of membrane, cell wall, and extracellular proteins were extremely valuable for a recent verification of signal peptide predictions for B. subtilis (179). Such data sets are now becoming available for S. aureus, as exemplified by studies on the membrane and cell wall proteomes of S. aureus Phillips (127) and the extracellular proteomes of S. aureus strains derived from the recently sequenced NCTC8325 and COL strains (208,209) (Fig. 2).…”

Complementary Analysis of the Vegetative Membrane Proteome of the Human Pathogen Staphylococcus aureus

“…This study provides a comparison of those approaches applied to the vegetative membrane proteome of S. aureus. Although analytical techniques were the focus of this work, we also report the largest validated set of membrane proteins of exponentially growing populations of this serious human pathogen to date (9,10). As demonstrated before, microorganisms do not express their entire set of proteins under certain growth conditions.…”

“…The proteome of S. aureus consists of ϳ2600 proteins (6, 7) of which more than one-quarter possess one or more transmembrane domains (TMDs) 1 according to TMHMM 2.0 (8). Although first insights into the membrane proteome of S. aureus could be delivered (9,10), our knowledge is still limited, and in particular the detection of proteins with multiple TMDs lags far behind. There are several special features of membrane proteins that render their analysis so difficult (11,12).…”