The Staphylococcus aureus secretome - TI Pharma

Chapter 5
incubated at 95ºC. Proteins were separated by SDS-PAGE using precast NuPage gels (Invitrogen) and
subsequently blotted onto a nitrocellulose membrane (Protran ® , Schleicher & Schuell). The presence of
IsaA, Aly or DsbA was monitored by immunodetection with specific polyclonal antibodies raised in
mice (IsaA, Aly) or rabbits (DsbA (Kouwen et al., 2007)) at 1:10.000 dilution. Bound primary
antibodies were visualized using fluorescent IgG secondary antibodies (IRDye 800 CW goat antimouse/anti-rabbit
from LiCor Biosciences). Membranes were scanned for fluorescence at 800 nm using
the Odyssey Infrared Imaging System (LiCor Biosciences).
Results
The exoproteomes of secG and secY2 mutant S. aureus strains
To investigate the roles of SecG and SecY2 in the biogenesis of the S. aureus exoproteome,
the respective genes were completely deleted from the chromosome of S. aureus strain
RN4220. This resulted in the single mutant strains ∆secG and ∆secY2, and the double mutant
∆secG ∆secY2. Next, cells of these mutants were grown in TSB medium until they reached
the stationary phase (Figure 1; not shown for the ∆secY2 strain).
Figure 1 Growth of S. aureus secG and secG secY2 mutants. The S. aureus strains RN4220 ∆secG (A), ∆secG
∆secY2 (B), and the parental strain RN4220 were grown in 100 ml TSB medium under vigorous shaking at 37 o C.
Sampling points for the preparation of extracellular proteins are indicated in the growth curve by arrows.
All three mutants displayed similar exponential growth rates as the parental strain. However,
the secG secY2 double mutant entered the stationary phase at a lower optical density
(OD540=8) than the parental strain and the ∆secG mutant (OD540=15). Extracellular proteins
were collected from the supernatant of the cell cultures that had reached stationary phase for
analysis by 2-D PAGE (Figures 1 and 2). Comparison of the exoproteomes of the secG
mutant and its parental strain revealed that eleven proteins with Sec-type signal peptides and
type I signal peptidase cleavage sites (i.e. SAOUHSC-00094, SdrD, Sle1, Geh, Hlb, HlY,
HlgB, HlgC, Plc, SAOUHC-02241 and SAOUHSC-02979) were present in significantly
decreased amounts when SecG was absent from the cells. This was also true for the secreted
moiety of the polytopic membrane protein YfnI, which is processed by signal peptidase I as
was previously shown for the YfnI homologue of B. subtilis (Antelmann et al., 2001). In
contrast, the amounts of three other exoproteins (i.e. IsaA, SsaA, and Spa) were considerably
increased due to the secG deletion (Figure 2A; Table 3A). These effects of the secG mutation
were fully compensated when secG was ectopically expressed from plasmid secG-pCN51
(Figure 2C). Northern blot analyses revealed similar transcript levels for geh, hlb and spa in
the secG mutant and the parental strain RN4220. This shows that the changes in the amounts
100