A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome

Abstract

The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen.

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

“…Probably a subpopulation could only be analyzed. This assumption is strengthened by previously monitored spreading of proteins over several sub-proteomes (30). Therefore the absolute quantification workflow has to be modified for membrane proteins as well as for small proteins.…”

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…Generation of Voronoi Treemaps-Voronoi treemaps have been proven as a powerful tool for the visualization of large proteomic data, mainly of S. aureus and B. subtilis (48,50). In order to visualize our complex data set, we functionally mapped the quantified L. pneumophila proteome based on its TIGR classification (51).…”

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

“…To obtain the total cytoplasmic proteins extracts as described by Becher et al (2009), 50 ml of culture was centrifuged (7000 g) for 10 min at 4 uC. Cell pellets were washed twice with 1 ml ice-cold TE buffer after resuspension in 1 ml TE buffer and transferred into screw-top tubes containing 500 ml glass beads.…”

“…For extraction of the secreted proteins, as described by Becher et al (2009), the supernatant of 50 ml culture volume was separated from cells by centrifugation (10 min, 7000 g, 4 uC) and transferred into a new tube. Then, 5 ml fresh pure TCA (final concentration 10 % (v/v)) was added to the supernatant and the proteins were precipitated overnight at 4 uC.…”

“…The protein pellet was dissolved in 8 M urea/2 M thiourea and mixed by shaking for 20 min at room temperature. The solution was finally centrifuged for 15 min at 21 000 g and the supernatant was transferred into a new microtube.To obtain the total cytoplasmic proteins extracts as described by Becher et al (2009), 50 ml of culture was centrifuged (7000 g) for 10 min at 4 uC. Cell pellets were washed twice with 1 ml ice-cold TE buffer after resuspension in 1 ml TE buffer and transferred into screw-top tubes containing 500 ml glass beads.…”