The Staphylococcus aureus secretome - TI Pharma

General summary and discussion
suggesting that this component of the secretion machinery is dispensable for host subversion
by S. aureus.
Chapter 6 of this thesis deals with the modification and processing of certain translocated
proteins by the class A sortase (SrtA) of S. aureus and S. epidermidis. SrtA attaches proteins
with an LPxTG motif to the cell wall, by cleaving this motif between the conserved Thr and
Gly residues and simultaneously attaching the Thr residue to peptidoglycan. In this study, the
extracellular proteins of sortase A mutants from S. aureus and S. epidermidis were analyzed.
Deletion of the srtA genes of these bacteria resulted in the dislocation of several LPxTG
proteins, such as ClfA, SasG, SdrC, SdrD, and protein A of S. aureus, and the Aap protein of
S. epidermidis 1457, from the cell wall to the growth medium. Nevertheless, substantial
amounts of these proteins remained cell wall-bound through non-covalent interactions
facilitated by cell wall-binding domains. The protein dislocation phenotypes of srtA mutations
in S. aureus and S. epidermidis were fully reversed by ectopic expression of srtA genes of
either species. Interestingly, the class C sortase (SrtC) from S. epidermidis 12228 was capable
of reversing the dislocation of ClfA, SasG and Aap to significant extents, showing that the
substrate specificities of SrtA and SrtC overlap at least partially. By contrast, SrtC was unable
to restore the covalent cell wall attachment of protein A and the SdrC and SdrD proteins in
the S. aureus srtA mutant. Interestingly, biofilm formation was affected in srtA mutants of S.
aureus, but not in the S. epidermidis srtA mutant. This difference can be correlated to the
expression levels of particular covalently cell wall-bound proteins involved in proteindependent
biofilm formation, such as S. aureus SasG and its S. epidermidis homologue Aap.
Remarkably, SrtA activity was a limiting determinant for protein-dependent biofilm
formation in S. aureus and S. epidermidis, whereas SrtC expression interfered with biofilm
formation in S. epidermidis 1457. Taken together, these findings imply that sortases can have
modulating roles in staphylococcal biofilm formation.
While the large mechano-sensitive membrane channel MscL can form pores that are large
enough to allow the passage of small proteins across the membrane, no evidence was obtained
that an S. aureus mscL mutant would be blocked in the release of certain proteins. In chapter
7, an interesting phenotype of the mscL mutant strain is however described. This relates to the
production of the extremely stable and broad-spectrum lantibiotic sublancin 168 by Bacillus
subtilis strain 168. Known sublancin 168 susceptible organisms include important pathogens,
such as S. aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has
remained elusive. The studies in chapter 7 were, therefore, aimed at the identification of
cellular determinants for bacterial susceptibility towards sublancin 168. Growth inhibition and
competition assays on plates and in liquid cultures revealed that sublancin 168-mediated
growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl
concentration in the growth medium. Added NaCl did not influence the production, activity or
stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells towards
this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells towards
sublancin 168 was shown to depend on the presence of MscL. These findings demonstrate
that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that
may either serve as a direct target for this lantibiotic, or as a gate of entry to the cytoplasm
both in B. subtilis and S. aureus.
The combination of bioinformatics and proteomics to investigate the secretome of S. aureus
has been proven to be a powerful approach for dissecting the functions of secretome
components. That this approach is useful not only for the analyses of pathogens, but also for
industrial workhorses like Bacillus licheniformis is shown in chapter 8. From the genome
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