The Staphylococcus aureus secretome - TI Pharma

Chapter 4
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O
540
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Time (h)
SH1000+
SH1000-
secY2+
secY2-
comGA
dsbA
lgt
prsA
Figure 4 Growth properties of mutant S. aureus strains and RNAIII production. (A) Growth curves of several
different S. aureus SH1000 secretion mutants. Strains were grown in TSB at 37 o C under vigorous shaking and the
OD 540 was measured at hourly intervals. The S. aureus SH1000+ and ∆secY2+ strains show hemolysin activity on
blood agar plates, while the SH1000- and ∆secY2- strains do not show this feature. (B) RNA was prepared from S.
aureus cells grown in TSB to optical densities at 540 nm of 1, 10 or 15. Serial dilutions of total RNA of S. aureus
RN4220 wild-type (WT) or ∆prsA (left panels), or S. aureus SH1000 wild-type (WT) or ∆prsA (right panels) were
blotted and cross linked onto a positively charged nylon membrane. The membrane-bound RNA was hybridized
with a digoxigenin labeled RNA probe complementary to RNAIII. Chemiluminescence signals were detected with a
LumiImager (Roche Diagnostics, Mannheim, Germany).
Since the growth to high density coincided with a non-hemolytic phenotype on blood agar
plates, we tested whether these strains might have accumulated agr mutations, leading to the
loss of the regulatory RNAIII. Indeed, dot blot analyses performed for the S. aureus SH1000
prsA mutant strain, for which this high density growth phenotype was first observed, revealed
the loss of RNAIII production (Figure 4B). In contrast, the S. aureus RN4220 prsA mutant
produced RNAIII at levels comparable to those of the parental strain (Figure 4B).
Figure 5. Agr-like phenotypes of S. aureus SH1000-derived secretion mutants. (A) The S. aureus SH1000
parental strain (WT), an SH1000 ∆cidA strain, an SH1000 ∆comGC strain, an SH1000 ∆dsbA strain, and an
SH1000 ∆prsA strain were grown on blood agar plates and incubated overnight at 37ºC. Hemolytic activity is
detectable as a halo around the streaked cells. (B) S. aureus SH1000 (WT), S. aureus SH1000 ∆cidA, S. aureus
SH1000 ∆comC, S. aureus SH1000 ∆comGA, S. aureus SH1000 ∆comGC, and S. aureus SH1000 ∆dsbA were
grown in TSB medium at 37 o C till the early stationary phase. Samples of extracellular proteins isolated from the
growth medium (M), non-covalently cell wall-bound proteins (CW) and total cells (C) were used for Western
blotting and immunodetection with specific antibodies against TrxA or SspB. Note that the antibodies against TrxA
are also bound by the IgG-binding proteins protein A (Spa) and Sbi. Bands corresponding to Spa, Sbi, SspB and
TrxA are marked with arrows.
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