In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325-4 and Newman. About one-third of the trapped proteins are previously shown to be unstable or to be substrates of ClpP in other bacteria, supporting the validity of the ClpP-TRAP. This group of proteins encompassed the transcriptional regulators CtsR and Spx, the ClpC adaptor proteins McsB and MecA, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp-proteolysis in a number of pathways that contribute to the success of S. aureus as a human pathogen.

In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.