In contrast hemolysin activity was reduced during sessile growth indicating that the deletion of secDF may have effects on overall metabolism. SpA seemed to be impaired PRN1371 in reaching its destined subcellular localization. In the

secDF mutant SpA accumulated in the membrane, was reduced in the cell wall fraction but was found in increased amounts in the supernatant. Altered secretion and processing of SpA might be due to impaired cell wall anchoring by the membrane protein sortase. However, Mazmanian et al. have shown that the extracellular enterotoxin B fused to the sorting signal of SpA accumulates in the cytoplasm and to a lesser extent in the membrane in a sortase mutant [48]. Thus, SpA might migrate by an alternate mechanism into the supernatant, circumventing linking to the peptidoglycan. A similar divergent effect on protein secretion

as we observed in the secDF mutant was found in a secG mutant. There SpA was found in increased amounts in the exoproteome, despite unaffected transcription [11]. In contrast, we found deletion of secDF to change mRNA levels for many of the analyzed genes, such as atl, coa, hla, hld and spa. GSK126 concentration The lack of secDF therefore seems to have a different impact on virulence factor expression than secG, influencing, most likely indirectly, transcription in addition to translocation. The absence of SecDF could especially cause a defective or reduced membrane insertion of sensor proteins belonging to one of the numerous S. aureus two component systems MTMR9 contributing to virulence

factor regulation and to adaptations to different growth conditions (reviewed in [49, 50]). The reduced hld levels in the mutant suggests that the secDF deletion affected at least one two component system by impairing signaling via the agr quorum sensor [51]. This study and the work of Sibbald et al. [11] once more demonstrate that protein and mRNA levels do not necessarily correlate. Specific regulation at the protein level has been shown for certain transcription factors in S. aureus [52, 53]. Such a control of protein stability via chaperones and proteases might exist as well for virulence factors. Interestingly, in E. coli, secY, yidC and secD mutants were shown to induce the Cpx system, which up-regulates the expression of factors involved in folding and proteolysis in response to abnormal proteins in the outer membrane, the periplasmic space or the plasma membrane [54]. The induction of similar systems in the S. aureus secDF mutant due to clogging of the membrane, as suggested by the increased amounts of SpA in this compartment, could be an additional factor influencing protein stability and lead to the partially incoherent mRNA and protein levels, as seen for hla, hld and spa during planktonic growth. Conclusions This work provides evidence that although secDF is dispensable in S. aureus, its deletion leads to a pleiotropic phenotype.