Identification ofStaphylococcus aureusexotoxins by combined sodium dodecyl sulfate gel electrophoresis and matrix-assisted laser desorption/ ionization-time of flight mass spectrometry

Abstract

Staphylococcus aureus is an important human pathogen whose pathogenesis involves the synthesis of cell wall associated virulence factors and secreted toxins with damaging effects on the host cells. Most of these pathogenic factors are synthesized in a growth-phase dependent manner as a response to environmental stress like heat, lack of nutrients or other deleterious conditions. Conventional identification of these pathogenic factors is based on Western blot analysis or enzyme-linked immunosorbent assay (ELISA) and is limited by the commercial availability of antibodies against these toxins. We report here the use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for monitoring the pathogenic factors of S. aureus. For the identification of pathogenic factors, a methicillin sensitive strain of S. aureus, ATCC-29213, was grown at 37 degrees C or 42 degrees C in brain-heart infusion broth and harvested during the early stationary phase of growth. Secreted proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzymatically digested with trypsin and analyzed by MALDI-TOF mass spectrometry. When grown at 42 degrees C, alpha- and beta-hemolysins were found to accumulate in S. aureus supernatants while the concentration of protein A was slightly decreased. The identity of some of these toxins was confirmed by Western-blot analysis. MALDI-TOF mass spectrometry combined with sodium dodecyl sulfate gel electrophoresis represents a rapid and simple approach to characterize the virulence of S. aureus strains which seems to be particularly valuable for the identification of S. aureus exotoxins for which ELISA is not established.

Antibacterial Effects of Green Tea Polyphenols on Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

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Proteomics of Protein Secretion by Bacillus subtilis : Separating the “Secrets” of the Secretome

“…Bernardo et al (13) performed a proteomic study aimed at the characterization of virulence of different S. aureus strains. Extracellular protein spots that were present in the medium of all tested strains were identified as protein A, hemolysins, lipases, and autolysins.…”

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“…There has been considerable effort directed toward defining the S. aureus secretome (9,14,17,36,37,54,55,71,79,99,(117)(118)(119). Some of the earliest efforts were computational approaches that relied on identifying conserved features of Sec-type N-terminal signal sequences.…”