Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 25

With this comprehensive proteome map, we have one essential tool for a better
understanding of cell physiology of the human pathogen, S. aureus .
Extracellular proteins constitute a reservoir of virulence factors and have
important roles in the pathogenicity of bacteria. Preliminary genome data show
that the various strains encode very different sets of virulence factors. In
addition to this genomic variability, differences in the activity of virulence-
associated regulators are believed to be responsible for variations in the
expression of some virulence factors in clinical isolates. A comprehensive
analysis of the extracellular proteome of S. aureus can show whether individual
virulence genes are (1) expressed and secreted, and if yes, (2) in which
quantities, and (3) under which environmental conditions. The expression of
virulence genes is regulated in a coordinated fashion during the growth cycle by
a complex network of regulators. The production of extracellular proteins takes
place mainly during the late exponential and post-exponential phase at high
cell densities, and at the same time, the synthesis of surface-associated proteins
is down-regulated (6) . The best characterized regulators of virulence gene
expression to date are Agr ( a ccessory g ene r egulator) and SarA ( s taphylococcal
a ccessory r egulator) (7) . We have compared the 2D pattern of extracellular
protein extracts of wild-type cells with the 2D patterns of the respective
regulatory mutants in order to identify proteins whose synthesis is influenced
by these regulatory genes (6,8) .
The secretome map is also a valuable tool for evaluating the fitness
and virulence of clinical isolates from patients with different diseases. The
comparison of the extracellular protein patterns revealed considerable hetero-
geneity between these strains, which might explain their different virulence
potential. To date, at least 90 of the proteins identified in the supernatant of
various S. aureus strains showed signal sequences typical for Sec-translocated
proteins. As expected, many of these proteins were already known to play a
role in the virulence of S. aureus (6,8,9,10) .
2. Materials
2.1. Cell Culture, Cell Lysis, and Protein Preparation
1. Tryptone soy broth (TSB) (Oxoid, Wesel, Germany), autoclaved for 15 min.
2. Luria-Bertani broth (LB) (Invitrogen, Karlsruhe, Germany) pH 7.5, autoclaved
for 20 min.
3. Basic medium (2×): Dissolve 3.54 g of Na 2 HPO 4 ,2.72gofKH 2 PO 4 , 0.4 g of
MgSO 4 7H 2 O,1gofNH 4 Cl, and 1 g of NaCl in 1 L deionized water. Autoclave
for 20 min.
4. 1 M citrate: Prepare in deionized water and autoclave for 20 min.
5. 20% (w/v) glucose: Prepare in deionized water and autoclave for 10 min.