The Staphylococcus aureus secretome - TI Pharma

Characterization of Staphylococcus aureus secretion mutants
The significant differences in the exoproteome of the secG mutant were highly unexpected
since deletion of secG in E. coli and B. subtilis does not seem to have a strong impact on
protein export in these bacteria (Nishiyama et al., 1994;Van Wely et al., 1999). As further
detailed in Chapter 5, many proteins are present at increased or decreased levels in the
exoproteome of the S. aureus RN4220 secG mutant and this effect was reversed by ectopic
expression of secG. Proteins that were detected in reduced amounts include well-known
secreted virulence factors, such as the lipase Geh, the alpha and beta hemolysins and the
leukocidins F and S, as well as the cell surface proteins SdrC and SdrD. Conversely, the
extracellular levels of other virulence factors, like protein A, the immunodominant antigen A
(IsaA) and the secretory antigen precursor SsaA were significantly increased. These
observations suggest that the absence of SecG changes the translocation efficiency of
different exoproteins via the SecYE channel to different extents. However, we cannot
completely rule out indirect effects on the transcription of the genes for certain exoproteins,
their translation, or their post-translocational folding or degradation. In contrast to the secG
mutation, no secretion defect was observed upon the deletion of the gene for the accessory
Sec channel component SecY2. This confirmed the results obtained by Siboo et al., who
showed that a secY2 mutation does not alter the S. aureus exoproteome (Siboo et al., 2008).
Till now, the only known SecY2-dependent protein of S. aureus is SraP, and it seems as if the
SecA2/SecY2 translocon is devoted to the translocation of this protein alone. Whether SecG
and/or SecE interact with this accessory SecA2/SecY2 translocon remains to be determined.
Similar to the secY2 deletion strain, no secretion defects were observed for strains lacking the
tatA and/or tatC genes, the comGA, comGC or comC genes, or the genes for the holin CidA
and antiholin LrgA (Figure 6B). This suggests that the Tat, Comand holing-antiholin
pathways serve very specific roles in the translocation of particular non-abundantly expressed
proteins, or proteins that are not detectable by 2-D PAGE due to their particular pI and size
properties. This view was recently confirmed for the S. aureus Tat pathway, which seems to
be required for the translocation of only one protein, namely FepB (Biswas et al.,
2009;Yamada et al., 2007). So far, we have not been able to detect FepB by our 2-D PAGE
approach.
The spsA gene encodes a type I signal peptidase that seems to lack the active site serine and
lysine residues (van Roosmalen et al., 2004). Interestingly, the occurrence of this type of an
apparently inactive type I signal peptidase is wide-spread amongst the Firmicutes.
Unfortunately, our proteomics studies did not shed light on the biological function of SpsA as
no differences were observed between the exoproteomes of our spsA deletion mutant and the
parental strain RN4220 (Figure 6).
The exoproteome of the lgt mutant showed the most pronounced differences compared to the
parental strain (Figure 6A). This result was not completely unexpected as previous studies of
Stoll et al. (Stoll et al., 2005) had shown that an S. aureus lgt mutant released high amounts of
certain unmodified prelipoproteins, such as the oligopeptide-binding protein OppA, the
peptidyl-prolyl cis-trans isomerase PrsA, and the staphylococcal iron transporter SitC, into the
growth medium. As shown by Western blotting, the same was true for the unmodified pre-
DsbA precursor, which was completely released into the growth medium of lgt mutant cells.
In contrast, in the parental strain, the mature DsbA fractionated with cells and non-covalently
cell wall-associated proteins (Figure 7). The latter finding seems to suggest that some mature
DsbA is not retained at the membrane but in the cell wall. These findings confirm earlier
observations of pre-lipoprotein release by lgt mutant strains of B. subtilis (Antelmann et al.,
2001;Tjalsma et al., 1999) and L. lactis (Venema et al., 2003). The release of lipoprotein
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