The Staphylococcus aureus secretome - TI Pharma

Chapter 5
Discussion
The extracellular and surface-associated proteins of bacterial pathogens, such as S. aureus,
represent an important reservoir of virulence factors. Accordingly, protein export mechanisms
will contribute to the virulence of these organisms. While protein export has been well
characterized in model organisms, such as E. coli and B. subtilis, relatively few functional
studies have addressed the protein export pathways of S. aureus. Notably, the Sec pathway is
generally regarded as the main pathway for protein export but, to date, this has not been
verified experimentally in S. aureus. Therefore, the present studies were aimed at assessing
the role of the Sec pathway in establishing the extracellular proteome of S. aureus. We
focused attention on the non-essential channel component SecG as this allowed a facile coassessment
of the non-essential accessory Sec channel component SecY2. Our results show
that the extracellular accumulation of proteins is affected to different extents by the absence
of SecG: some proteins are present in reduced amounts, some are not affected and some are
present in elevated amounts. Furthermore, the effects of the absence of SecG are exacerbated
by deletion of SecY2, suggesting that SecY2 directly or indirectly influences the functionality
of the general Sec pathway. This is all the more remarkable since the absence of SecY2 by
itself had no detectable effects on the composition of the extracellular proteome of S. aureus.
The observation that the secretion of a wide range of proteins was affected by the
absence of SecG is consistent with the fact that all of these proteins contain Sec-type signal
peptides.On the other hand, this finding is remarkable since studies in other organisms, such
as E. coli (Nishiyama et al., 1993) and B. subtilis (Van Wely et al., 1999) have shown that
deletion of secG had fairly moderate effects on protein secretion in vivo. In B. subtilis, a
phenotype of the secG mutation was only observed under conditions of high overproduction
of secretory proteins (Van Wely et al., 1999). Clearly, our present data show that SecG is
more important for Sec-dependent protein secretion in S. aureus than in B. subtilis or E. coli.
Importantly, the transcription of genes for three proteins (Geh, Hlb and Spa) that were
affected in major ways by the absence of SecG was not changed, and all observed effects of
the secG mutation could be reversed by ectopic expression of secG. This suggests that the
observed changes in the exoproteome composition of the S. aureus secG mutant strain relate
to changes in the translocation efficiency of proteins through the Sec channel rather than
regulatory responses at the gene expression level. This could be due to altered recognition of
the respective signal peptides or mature proteins by the SecG-less Sec channel, or
combinations thereof. However, some indirect effects, for example at the level of translation
of exported proteins or cell wall binding of proteins like IsaA, LytM, Spa and SsaA, can
currently not be excluded especially since no proteins were found to accumulate inside the
secG mutant cells (data not shown). It remains to be shown why the extracellular
accumulation of particular proteins is affected by the absence of SecG, while that of other
proteins remains unaffected.
Unexpectedly, our studies revealed that export of the IgG-binding protein Sbi to the
cell wall was almost completely blocked in secG mutant strains. The reason why this export
defect was not detected by 2-D PAGE relates to the fact that Sbi is predominantly cell wallbound
in the tested S. aureus strains under the experimental conditions used. It has been
proposed previously that Sbi would remain cell wall-attached through a proline-rich wallbinding
domain and electrostatic interactions (Zhang et al., 1998). Nevertheless, Burman and
colleagues showed that Sbi is extracellular and they suggested that cell surface-bound Sbi
might be disadvantageous for the bacterium due to its role in modulating the complement
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