title: Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus.
abstract: 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.
authors.affiliation: Department of Veterinary Disease Biology, University of Copenhagen, Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark.
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Feng
authors.forename: Stephan
authors.initials: S
authors.lastname: Michalik
authors.forename: Anders N
authors.initials: AN
authors.lastname: Varming
authors.forename: Julie H
authors.initials: JH
authors.lastname: Andersen
authors.forename: Dirk
authors.initials: D
authors.lastname: Albrecht
authors.forename: Lotte
authors.initials: L
authors.lastname: Jelsbak
authors.forename: Stefanie
authors.initials: S
authors.lastname: Krieger
authors.forename: Knut
authors.initials: K
authors.lastname: Ohlsen
authors.forename: Michael
authors.initials: M
authors.lastname: Hecker
authors.forename: Ulf
authors.initials: U
authors.lastname: Gerth
authors.forename: Hanne
authors.initials: H
authors.lastname: Ingmer
authors.forename: Dorte
authors.initials: D
authors.lastname: Frees
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial; D024881:Drug Resistance, Bacterial / Q000235:genetics; D049071:Endopeptidase Clp / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D018869:Heat-Shock Response / Q000235:genetics; D046915:Penicillin-Binding Proteins; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C118338:CtsR protein, bacteria; D003598:Cytoskeletal Proteins; D004269:DNA, Bacterial; C071525:FtsZ protein, Bacteria; D046915:Penicillin-Binding Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C487789:mecA protein, Staphylococcus aureus; C122138:peroxide repressor proteins; D011494:Protein Kinases; D049071:Endopeptidase Clp; D010444:Peptide Elongation Factor Tu