The Staphylococcus aureus secretome - TI Pharma

Chapter 4
assay, 25 L4-stage nematodes were transferred to the plate containing the S. aureus spots, and each
assay was performed in triplicate. The plates were incubated at 25°C and the numbers of living
nematodes were counted at 24 hour intervals. Nematodes that were not moving after plate tapping or
gentle touching with a platinum wire were counted as dead. Statistical analysis of nematode survival
was performed using the StatView program version 5.0 (SAS Institute Inc.) to create the cumulative
survival plots by the Kaplan-Meier method.
Results and Discussion
Construction of an S. aureus secretion mutant collection
Judged by previous studies in organisms like E. coli and B. subtilis, at least 30 proteins can
fulfill potential roles in protein secretion by S. aureus (Table 1). Of these 30 proteins, the Ffh,
FtsY, SecA1, SecY1, SecE and SpsB proteins are known to be essential for growth of E. coli,
B. subtilis and most likely also S. aureus (Ji et al., 2001;Cregg et al., 1996;Sibbald et al.,
2006). Furthermore, in these organisms the role of YrbF in protein secretion seemed so far of
minor importance. We therefore focused our attention on the potential roles of the remaining
23 proteins in protein secretion. To this purpose, we tried to delete the corresponding genes
with the pMAD chromosomal integration-excision system (Figure 2). In this manner, we were
able to completely delete the secY2, secG, lgt, spsA, lspA, prsA, dsbA, srtA, srtB, tatA, tatC,
comGA, comGC, comC, cidA, or lrgA genes from the S. aureus RN4220 chromosome. In
contrast, several attempts to delete the secA2, secDF, spoIIIJ/yqjG, esaA, essA, essB and essC
genes remained unsuccessful, as was the case for a control experiment in which we tried to
delete spsB. The observation that secDF and spoIIIJ/yqjG could not be deleted is consistent
with the results of a recent Transposon-Mediated Differential Hybridisation (TMDH) analysis
in which about a million transposon mutants were screened using a microarray approach
(Chaudhuri et al., 2009). This analysis revealed 351 S. aureus genes that are of major
importance for growth and cell viability, among which the secDF and spoIIIJ/yqjG genes.
This finding points towards an important difference in the secretion machinery of B. subtilis
and S. aureus since secDF is completely dispensable for growth, cell viability and protein
secretion in B. subtilis (Bolhuis et al., 1998), and the same is true for the individual spoIIIJ
and yqjG genes (Tjalsma et al., 2003). However, consistent with the situation in S. aureus, the
B. subtilis spoIIIJ and yqjG genes cannot be deleted simultaneously, which shows that YidC
function is essential for this organism (Tjalsma et al., 2003). Furthermore, the studies by
Chaudhuri et al. confirmed the essentiality of the ffh, ftsY, secA1, secE, secY1 and spsB genes
(Chaudhuri et al., 2009). Interestingly, the secA2, esaA, essA, essB and essC genes were not
identified as potentially essential in the TMDH analysis. Studies by Burts et al. (Burts et al.,
2005;Burts et al., 2008) have shown that the esaA, essA, essB and essC genes can be mutated,
which suggests that our attempts to delete these genes were unsuccessful due to an unknown
technical problem. However, we can presently not exclude the possibility that these genes are
essential in S. aureus RN4220 while being dispensable in other strains. Direct evidence that
secA2 is dispensable for S. aureus was provided by Siboo et al. (Siboo et al., 2008), who
reported the deletion of the secA2 gene from S. aureus ISP479C. This suggests that secA2 is
either essential in S. aureus RN4220 or that we were unlucky in our attempts to delete this
gene. As a final approach, we therefore attempted to deplete the cells of SecA2 by replacing
the original promoter sequences with the Pspac promoter through a single cross-over
integration of plasmid pMUTIN4 in front of the secA2 gene. As can be expected for a non-
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