The Staphylococcus aureus secretome - TI Pharma

Synthetic effects of secG and secY2 mutations on exoproteome biogenesis in
Staphylococcus aureus
Impaired export of cell wall-bound Sbi in secG mutant cells
Western blotting experiments were performed to investigate whether particular protein export
defects of the secG and secY2 mutants had remained unnoticed in the proteomic analyses.
These analyses included secreted proteins in the growth medium, non-covalently cell wall
attached and cellular proteins of S. aureus strains RN4220 and S. aureus SH1000.
Furthermore, we used specific antibodies against membrane proteins, lipoproteins, cell wall
proteins and exoproteins. For most tested proteins no differences were detectable between the
secG and/or secY2 mutant strains and their parental strain. However, these analyses showed
that a band of ~50 kDa, which was cross-reactive with all tested sera, had disappeared from
the fraction of non-covalently bound cell wall proteins of the secG mutant. It is known that
proteins, such as protein A (Sasso et al., 1991) and Sbi (Zhang et al., 1998) have IgG-binding
properties. To investigate whether the missing band would relate to protein A or Sbi, protein
fractions from a spa mutant, and a spa sbi double mutant, were included in the Western
blotting analyses.
Figure 4. Sbi localization to the cell wall of S. aureus depends on SecG. (A) S. aureus SH1000 (WT), S. aureus
SH1000 ∆secG, and S. aureus SH1000 ∆secG secG-pCN51 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 wallbound
proteins (CW) and total cells (C) were used for Western blotting and immunodetection with serum of mice
immunized with IsaA. As a contol for Sbi production, the strains S. aureus Newman ∆spa and S. aureus Newman
∆spa ∆sbi were included in the analyses. (B) Proteins of S. aureus SH1000 (wt), S. aureus SH1000 ∆secG, and S.
aureus SH1000 ∆secY2 were used for Western blotting and immunodetection as in (A). The position of Sbi is
marked with an arrow.
As shown in Figure 4A, the band of ~50 kDa that was missing from the non-covalently bound
cell wall proteins in the secG mutant was also missing from these proteins in the spa sbi
double mutant, but not in the spa single mutant (only the results for S. aureus SH1000 are
shown but essentially the same results were obtained for S. aureus RN4220). Taken together,
these findings show that Sbi is non-covalently bound to the cell wall of S. aureus RN4220 and
SH1000, and that SecG is required for export of Sbi from the cytoplasm to the cell wall. As
was the case for the secreted S. aureus proteins detected by proteomics, Sbi export to the cell
wall was not affected by the absence of SecY2 (Figure 4B). Finally, it is noteworthy that Sbi
is only detectable amongst the non-covalently bound cell wall proteins of S. aureus RN4220
and SH1000, whereas it is detectable both in a cell wall-bound and a secreted state in S.
aureus Newman.
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