Relative quantitative comparisons of the extracellular protein profiles of Staphylococcus aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants using one-dimensional polyacrylamide gel electrophoresis and nanocapillary liquid chromatography coupled with tandem mass spectrometry

Abstract

One-dimensional polyacrylamide gel electrophoresis followed by nanocapillary liquid chromatography coupled with mass spectrometry was used to analyze proteins isolated from Staphylococcus aureus UAMS-1 after 3, 6, 12, and 24 h of in vitro growth. Protein abundance was determined using a quantitative value termed normalized peptide number, and overall, proteins known to be associated with the cell wall were more abundant early on in growth, while proteins known to be secreted into the surrounding milieu were more abundant late in growth. In addition, proteins from spent media and cell lysates of strain UAMS-1 and its isogenic sarA, agr, and sarA agr regulatory mutant strains during exponential growth were identified, and their relative abundances were compared. Extracellular proteins known to be regulated by the global regulators sarA and agr displayed protein levels in accordance with what is known regarding the effects of these regulators. For example, cysteine protease (SspB), endopeptidase (SspA), staphopain (ScpA), and aureolysin (Aur) were higher in abundance in the sarA and sarA agr mutants than in strain UAMS-1. The immunoglobulin G (IgG)-binding protein (Sbi), immunodominant staphylococcal antigen A (IsaA), IgG-binding protein A (Spa), and the heme-iron-binding protein (IsdA) were most abundant in the agr mutant background. Proteins whose abundance was decreased in the sarA mutant included fibrinogen-binding protein (Fib [Efb]), IsaA, lipase 1 and 2, and two proteins identified as putative leukocidin F and S subunits of the two-component leukotoxin family. Collectively, this approach identified 1,263 proteins (matches of two peptides or more) and provided a convenient and reliable way of identifying proteins and comparing their relative abundances.

FIG. 1.

A representative growth curve as determined spectrophotometrically of S. aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants grown in TSB at 37°C with rotary aeration (180 rpm). S. aureus UAMS-1 (•), sarA (○), agr (▾), and sarA agr (▵) mutants are shown.

FIG. 2.

(a) A representative gel of a 1D SDS-PAGE analysis of spent media isolated from S. aureus UAMS-1 after 3, 6, 12, and 24 h of growth in TSB at 37°C with rotary aeration at 180 rpm. (b) A representative gel of a 1D SDS-PAGE analysis of spent media isolated from S. aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants after 3 h of growth in TSB at 37°C with rotary aeration at 180 rpm. (c) A representative gel of a 1D SDS-PAGE analysis of cell lysates isolated from S. aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants after 3 h of growth in TSB at 37°C with rotary aeration at 180 rpm. The positions of molecular mass markers in lanes M (in kilodaltons) are shown to the left of the gels.

FIG. 3.

Normalized peptide number of a select group of known virulence factors (a), known cytolytic proteins (b), known proteases (c), and putative exported proteins (d) detected in spent media isolated from S. aureus UAMS-1 after 3, 6, 12, and 24 h of growth.

FIG. 3.

Normalized peptide number of a select group of known virulence factors (a), known cytolytic proteins (b), known proteases (c), and putative exported proteins (d) detected in spent media isolated from S. aureus UAMS-1 after 3, 6, 12, and 24 h of growth.

FIG. 4.

Normalized peptide number of a select group of cell wall-associated proteins detected in spent media isolated from S. aureus UAMS-1 after 3, 6, 12, and 24 h of growth.

FIG. 5.

NPN of a select group of known extracellular proteins detected in spent media isolated from S. aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants after 3 h of growth.

FIG. 6.

NPN of known cell wall-associated proteins detected in spent media isolated from S. aureus UAMS-1 and its sarA, agr, and sarA agr regulatory mutants after 3 h of growth.