Identification of the Putative Staphylococcal AgrB Catalytic Residues Involving the Proteolytic Cleavage of AgrD to Generate Autoinducing Peptide
2005; Elsevier BV; Volume: 280; Issue: 17 Linguagem: Inglês
10.1074/jbc.m411372200
ISSN1083-351X
AutoresRongde Qiu, Wuhong Pei, Linsheng Zhang, Jianqun Lin, Guangyong Ji,
Tópico(s)Antimicrobial Peptides and Activities
ResumoThe P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic α-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria. The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic α-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria. The Agr quorum sensing system encoded by the staphylococcal accessory gene regulator (agr) 1The abbreviations used are: agr, accessory gene regulator; AIP, auto-inducing peptide; BlaZ, β-lactamase; Pbla, bla promoter; SMM, sucrose-sodium maleate-MgCl2; GFP, green fluorescent protein; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; TPCK, Nα-tosyl-l-phenylalanine chloromethyl ketone; VFK-CMK, d-Val-Phe-Lys chloromethyl ketone; E-64, 1-trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane; DSP, 3,3′-dithiobis(succinimidyl propionate); nt, nucleotide(s); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: agr, accessory gene regulator; AIP, auto-inducing peptide; BlaZ, β-lactamase; Pbla, bla promoter; SMM, sucrose-sodium maleate-MgCl2; GFP, green fluorescent protein; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; TPCK, Nα-tosyl-l-phenylalanine chloromethyl ketone; VFK-CMK, d-Val-Phe-Lys chloromethyl ketone; E-64, 1-trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane; DSP, 3,3′-dithiobis(succinimidyl propionate); nt, nucleotide(s); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. is one of the two-component signal transduction systems that involved in the regulation of virulence gene expression (1Novick R.P. Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (1000) Google Scholar, 2Novick R.P. Muir T.W. Curr. Opin. Microbiol. 1999; 2: 40-45Crossref PubMed Scopus (130) Google Scholar, 3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google Scholar). The agr locus consists of two major transcripts: RNAII and RNAIII, that are transcribed divergently from the two agr promoters, P2 and P3, respectively (3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google Scholar). RNAII encodes four genes (agrA, -B, -C, and -D) whose products constitute a quorum sensing system: AgrC and AgrA are the sensor kinase and the response regulator of the Agr two-component signal transduction system, respectively (3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google Scholar, 4Koenig R.L. Ray J.L. Maleki S.J. Smeltzer M.S. Hurlburt B.K. J. Bacteriol. 2004; 186: 7549-7555Crossref PubMed Scopus (158) Google Scholar); AgrD is the propeptide of the autoinducing peptide (AIP) that is secreted from the bacteria (5Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (512) Google Scholar) and functions as the ligand for AgrC (6Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (193) Google Scholar); and AgrB is a membrane protein that is involved in the processing of AgrD (7Zhang S. Stewart G.C. J. Bacteriol. 2000; 182: 2321-2325Crossref PubMed Scopus (28) Google Scholar). AgrC is a membrane protein with its N-terminal half integrated into the cytoplasmic membrane that contains the AIP binding site (6Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (193) Google Scholar, 8Lyon G.J. Wright J.S. Muir T.W. Novick R.P. Biochemistry. 2002; 41: 10095-10104Crossref PubMed Scopus (169) Google Scholar), and with its C-terminal half located in the cytoplasm that possesses histidine kinase activity (6Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (193) Google Scholar). Among the identified AgrCs so far from various species of staphylococci, the N-terminal halves are divergent and the C-terminal halves are highly conserved (9Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (624) Google Scholar, 10Dufour P. Jarraud S. Vandenesch F. Greenland T. Novick R.P. Bes M. Etienne J. Lina G. J. Bacteriol. 2002; 184: 1180-1186Crossref PubMed Scopus (165) Google Scholar). This reflects the fact that the AgrCs are activated only by their cognate AIPs but are inhibited by heterologous AIPs (9Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (624) Google Scholar, 11Lyon G.J. Wright J.S. Christopoulos A. Novick R.P. Muir T.W. J. Biol. Chem. 2002; 277: 6247-6253Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Mayville P. Ji G. Beavis R. Yang H. Goger M. Novick R.P. Muir T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1218-1223Crossref PubMed Scopus (384) Google Scholar, 13Otto M. Sussmuth R. Jung G. Gotz F. FEBS Lett. 1998; 424: 89-94Crossref PubMed Scopus (127) Google Scholar, 14Jarraud S. Lyon G.J. Figueiredo A.M. Gerard L. Vandenesch F. Etienne J. Muir T.W. Novick R.P. J. Bacteriol. 2000; 182: 6517-6522Crossref PubMed Scopus (244) Google Scholar, 15McDowell P. Affas Z. Reynolds C. Holden M.T. Wood S.J. Saint S. Cockayne A. Hill P.J. Dodd C.E. Bycroft B.W. Chan W.C. Williams P. Mol. Microbiol. 2001; 41: 503-512Crossref PubMed Scopus (148) Google Scholar). Based on the AIP cross-activation and cross-inhibition activities, four specificity groups of Staphylococcus aureus (9Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (624) Google Scholar, 14Jarraud S. Lyon G.J. Figueiredo A.M. Gerard L. Vandenesch F. Etienne J. Muir T.W. Novick R.P. J. Bacteriol. 2000; 182: 6517-6522Crossref PubMed Scopus (244) Google Scholar) and three groups of Staphylococcus epidemidis (10Dufour P. Jarraud S. Vandenesch F. Greenland T. Novick R.P. Bes M. Etienne J. Lina G. J. Bacteriol. 2002; 184: 1180-1186Crossref PubMed Scopus (165) Google Scholar, 16Donvito B. Etienne J. Greenland T. Mouren C. Delorme V. Vandenesch F. FEMS Microbiol. Lett. 1997; 151: 139-144Crossref PubMed Google Scholar, 17Van Wamel W.J. van Rossum G. Verhoef J. Vandenbroucke-Grauls C.M. Fluit A.C. FEMS Microbiol. Lett. 1998; 163: 1-9Crossref PubMed Scopus (42) Google Scholar) have been identified. Upon the binding of AIP, AgrC is autophosphorylated (6Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (193) Google Scholar). It has been proposed that the phosphoryl group of the phosphorylated AgrC is transferred to AgrA, and the phosphorylated AgrA then interacts with the P2 and P3 promoters to activate the transcription of both RNAII and RNAIII (3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google Scholar, 4Koenig R.L. Ray J.L. Maleki S.J. Smeltzer M.S. Hurlburt B.K. J. Bacteriol. 2004; 186: 7549-7555Crossref PubMed Scopus (158) Google Scholar, 6Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (193) Google Scholar). RNAIII is the actual regulator that activates the expression of genes encoding secreted virulence factors and represses those encoding cell surface-associated proteins (1Novick R.P. Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (1000) Google Scholar, 18Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (825) Google Scholar). AgrD is a membrane protein anchored in the inner leaflet of the cytoplasmic membrane via an amphipathic α-helix formed by its N-terminal region (19Zhang L. Lin J. Ji G. J. Biol. Chem. 2004; 279: 19448-19456Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). AgrD sequences from various staphylococcal species are remarkably divergent with only 4 identical amino acids (1Novick R.P. Mol. Microbiol. 2003; 48: 1429-1449Crossref PubMed Scopus (1000) Google Scholar). The mature AIPs isolated so far from a number of staphylococcal species are 7 to 9 amino acids in length, and all are thiolactone molecules containing a 5-amino acid ring linked by a thioester bond formed between the sulfhydryl group of a conserved cysteine residue and the carboxyl group of the C-terminal amino acid, except the Staphylococcus intermedius AIP, a lactone molecule that contains a ester bond formed between the hydroxyl group of a serine residue (in place of the cysteine residue that is absolutely conserved among other AIPs) and the carboxyl group of the C-terminal residue (11Lyon G.J. Wright J.S. Christopoulos A. Novick R.P. Muir T.W. J. Biol. Chem. 2002; 277: 6247-6253Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Mayville P. Ji G. Beavis R. Yang H. Goger M. Novick R.P. Muir T.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1218-1223Crossref PubMed Scopus (384) Google Scholar, 15McDowell P. Affas Z. Reynolds C. Holden M.T. Wood S.J. Saint S. Cockayne A. Hill P.J. Dodd C.E. Bycroft B.W. Chan W.C. Williams P. Mol. Microbiol. 2001; 41: 503-512Crossref PubMed Scopus (148) Google Scholar, 20Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google Scholar, 21Kalkum M. Lyon G.J. Chait B.T. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2795-2800Crossref PubMed Scopus (99) Google Scholar). The AIP sequence is in the middle of the AgrD sequence that is preceded by the N-terminal amphipathic α-helix and followed by a highly hydrophilic C-terminal region. The processing of AgrD to generate mature AIP involves the proteolytic cleavages at two processing sites, the thioester (or ester) bond formation, and the secretion of the mature AIP. AgrB is a membrane protein with six transmembrane segments including four transmembrane α-helices and two highly hydrophilic regions (22Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Like AgrD, the AgrBs sequenced from various staphylococcal species are also divergent, except the N-terminal region located in the cytoplasm and the two highly hydrophilic regions that are proposed to be in the membrane according to the AgrB topological model (22Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Computer analyses show similar hydropathy profiles among the AgrB sequences (22Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). It is likely that all AgrBs are structurally and functionally similar and the mechanisms of processing AgrD and of secreting the mature AIP by AgrBs are the same or similar even though the AgrD propeptides are different and the interaction between AgrB and AgrD is specific (23Zhang L. Ji G. J. Bacteriol. 2004; 186: 6706-6713Crossref PubMed Scopus (33) Google Scholar). In this study, we identified two amino acid residues in both S. aureus and S. intermedius AgrBs that were involved in the proteolytic cleavages of AgrD, and proposed that the staphylococcal AgrB protein was a putative cysteine endopeptidase. Bacterial Strains and Growth Conditions—S. aureus RN6390B is a derivative of NCTC8325 and is our standard laboratory group I strain. RN6911 is a derivative of RN6390B in which the agr locus is replaced by the tetM gene (18Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (825) Google Scholar). SA502A is our standard S. aureus group II strain (9Ji G. Beavis R. Novick R.P. Science. 1997; 276: 2027-2030Crossref PubMed Scopus (624) Google Scholar) and GJ2035 contains plasmid pI254 carrying an inducible bla operon in RN6911 (22Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). S. intermedius ATCC29663 was obtained from the American Type Culture Collection (Manassas, VA). Escherichia coli JM109 was used for cloning and BL21(DM3) was used for protein expression. The plasmids used in this study are listed in Table I. S. aureus cells were grown in CYGP broth (24Novick R.P. Methods Enzymol. 1991; 204: 587-636Crossref PubMed Scopus (468) Google Scholar), supplemented with either 5 μg/ml chloramphenicol or 5 μg/ml erythromycin or both when necessary. E. coli cells were grown in LB medium (25Sambrook J. W. R.D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Cell growth was monitored with a Klett-Summerson colorimeter with a green (540 nm) filter (Klett, Long Island City, NY). S. aureus cells expressing AgrB or AgrD or both under the control of the staphylococcal Pbla promoter were induced with 0.5 μg/ml methicillin. E. coli cells expressing the Agr protein(s) under the T7lac promoter control were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside.Table IStrains and plasmids used in this studyGenotype and descriptionRef.PlasmidspET11aE. coli T7 tag vector with T7lac promoterNovagenpGEM-TE. coli PCR product cloning vectorPromegapRN5543Staphylococcal cloning vector18Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (825) Google ScholarpRN5548Staphylococcal expression vector carrying a staphylococcal inducible Pbla promoter18Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (825) Google ScholarpRN6441Staphylococcal cloning vector18Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (825) Google ScholarpRN6683S. aureus group I agr P3-blaZ fusion3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google ScholarpRN6913S. aureus group I agrD in pRN55485Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (512) Google ScholarpLZ2003S. aureus RN6390B agrB in pRN644122Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google ScholarpLZ2004S. aureus RN6390B agrB-His6 in pRN554822Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google ScholarpLZ4012S. aureus RN6390B agrD(tldh) in pRN554819Zhang L. Lin J. Ji G. J. Biol. Chem. 2004; 279: 19448-19456Abstract Full Text Full Text PDF PubMed Scopus (53) Google ScholarpLZ5001S. aureus RN6390B agrB-His6 in pRN6441This studypLZ5002agrB(ΔN), deletion of the N-terminal 34 amino acids in pLZ2004This studypLZ5003agrB(ΔBsi), residues 73-78 AHGAHA changed to HHVHHH in pRN6912This studypLZ5004agrB(ΔPst), residues 123-125 YAP changed to AAA in pLZ2004This studypLZ5005agrB(ΔPst1), Y123A in pLZ2004This studypLZ5006agrB(ΔPst2), K129A/K130A in pLZ2004This studypLZ5007agrB(ΔPst3), Y123A/ K129A/K130A in pLZ2004This studypLZ5008agrB(C84S) in pRN6441This studypLZ5009agrB(H77A) in pRN6441This studypLZ5010agrB(H74A) in pRN6441This studypLZ5011agrB(S80A) in pRN6441This studypLZ5012agrB(S81A) in pRN6441This studypWP1002S. intermedius promoter P3-gfp fusion in pRN5543This studypWP1003gfp gene in pRN5543This studypWP1004S. intermedius promoter P3-blaZ fusion in pRN554320Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google ScholarpWP1102S. intermedius agrB-Si and S. intermedius agrD-Si in pRN554820Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google ScholarpWP1103T7 epitope-agrD-Si-His6 (TD-SiH) in pRN554820Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google ScholarpWP1104agrB-Si and TD-SiH in pRN554820Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google ScholarpWP1105agrB-Si(H77A) in pWP1102This studypWP1110agrB(chimeric agrB) in pWP1104This studypWP1111agrB-Si(D5A) in pWP1104This studypWP1112agrB-Si(N20A) in pWP1104This studypWP1113agrB-Si(H23A) in pWP1104This studypWP1114agrB-Si(R30A) in pWP1104This studypWP1115agrB-Si(Q34A) in pWP1104This studypWP1116agrB-Si(N39A) in pWP1104This studypWP1117agrB-Si(K42A) in pWP1104This studypWP1118agrB-Si(H77A) in pWP1104This studypWP1119agrB-Si(S81A) in pWP1104This studypWP1120agrB-Si(C84A) in pWP1104This studypWP1201T7 epitope-agrD-Si-His6 (TD-SiH) in pET11aThis studypWP1202agrB-Si-TD-SiH in pET11aThis study Open table in a new tab Plasmid Constructions—Plasmid pLZ5001 was constructed by cloning a ClaI DNA fragment containing the S. aureus group I agrB-His6 gene under the control of the Pbla promoter from pLZ2004 (22Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) into the ClaI site of pRN6441. pLZ5002 was made by ligating two NcoI-digested PCR products amplified from pLZ2004 using T4 polynucleotide kinasephosphorylated primers: GJ number 50, 5′-GTCTTAGCTAAAAATATAGG-3′ (in agrB, nt 1878–1897 of the S. aureus group I agr, agr-1; GenBank™ accession number X52543) and GJ number 45, 5′-GTAAATGAAGTCCATGGAATAATAG-3′ (around the NcoI site of pRN5548), and primers GJ number 51, 5′-CAATTTTACACCACTCTCCTC-3′ (in agrB, nt 1758–1778 in agr-1) and GJ number 44 5′-CTATTATTCCATGGACTTCATTTAC-3′ (complementary to GJ number 45), respectively. pLZ5003 was constructed the same way as pLZ5002 except the primer pairs used were GJ number 58 5′-AAGTGCACCATCACCCTTCTTCTTTTTGGT-3′ (in agrB, nt 1996–2025 in agr-1; changed nt, underlined) and GJ number 45, and GJ number 59 5′-AAGTGCACATGATGATGTCTTCTTATTAAATAAAAT-3′ (in agrB, nt 1970–2005 in agr-1; changed nt, underlined) and GJ number 44, and the PCR products were digested with HgiAI/NcoI. Plasmids pLZ5004 to pLZ5006 were constructed by ligating an appropriate NcoI/PstI DNA fragment of pLZ2004 and a NcoI/PstI-digested PCR product generated from pLZ2004 using primers GJ number 61 5′-AACTGCAGCAGCAGCTACTGAGATTACACCTAAAG-3′ (in agrB, nt 2122–2154 in agr-1; changed nt, underlined; PstI site, italic) and GJ number 44 (for pLZ5004), or LZ number 22 5′-GTTGCTGCAGGAGCAGCTACTGAGAT-3′ (in agrB, nt 2133–2158 in agr-1; changed nt, underlined; PstI site, italic) and GJ number 44 (for pLZ5005), or LZ number 23 5′-TGCTCCTGCAGCAACTGCTGCTAAGCCCAT-3′ (in agrB, nt 2144–2172 in agr-1; changed nt, underlined; PstI site, italic) and GJ number 45 (for pLZ5006). pLZ5007 was constructed the same way as pLZ5002 except the primer pairs used were LZ number 22 and GJ number 44, and LZ number 23 and GJ number 45, and the PCR products were digested with both PstI and NcoI. pLZ5008 was constructed by ligating two NcoI-digested PCR products amplified from pLZ2004 using primers LZ number 24 5′-TTGGTCTTATGTAGAAAGTATTATACT-3′ (in agrB, nt 2021–2047 in agr-1; changed nt, underlined) and GJ number 45, and LZ number 25 5′-AAAGAAGAAGGTGCATGTGCAC-3′ (in agrB, nt 1999–2020 in agr-1) and GJ number 44, followed by cloning a ClaI fragment of the resulting plasmid into the ClaI site of pRN6441. pLZ5009 was made by ligating a NcoI-digested PCR product generated by using oligonucleotides LZ number 21 5′-TGCAGCAGCACCTTCTTCTTTTTGGT-3′ (in agrB, nt 2000–2025 in agr-1; changed nt, underlined) and GJ number 45 as primers and pLZ2004 as a template, and an NcoI/BsiHKAI (the BsiHKAI site was blunted with T4 DNA polymerase) DNA fragment of pLZ2004, followed by cloning a ClaI fragment of the resulting plasmid into the ClaI site of pRN6441. To make pLZ5010 to pLZ5012, a HgiAI/NcoI-digested PCR product amplified from pLZ2004 was ligated with an appropriate HgiAI/NcoI fragment of pLZ2004, and a ClaI fragment carrying the mutated agrB was then cloned into the ClaI site of pRN6441l. The primer pairs used were H74A, 5′-GCATGTGCACCAGCTGCATGTCTTCT-3′ (in agrB, nt 1983–2008 in agr-1; changed nt, underlined) and GJ number 44 (for pLZ5010); S80A, 5′-ATGGTGCACATGCACCTGCTTCTT-3′ (in agrB, nt 1996–2019 in agr-1; changed nt, underlined), and GJ number 45 (for pLZ5011), and S81A 5′-ATGGTGCACATGCACCTTCTGCTTTTTG-3′ (in agrB, nt 1996–2023 in agr-1; changed nt, underlined) and GJ number 45 (for pLZ5012). Plasmid pWP1002 was constructed as follows: the putative P2/P3 promoter region of S. intermedius (20Ji G. Pei W. Zhang L. Qiu R. Lin J. Benito Y. Lina G. Novick R.P. J. Bacteriol. 2004; (in press)PubMed Google Scholar) was amplified by PCR using oligonucleotides WP12, 5′-CCATCACCAATGTGATGATG-3′ (P3 promoter region), and SINT11, 5′-CGGCTCTCCTCCTTGTTT-3′ (P2 promoter region), as primers and chromosomal DNA as template. The PCR product was cloned into an E. coli cloning vector pGEM-T (Promega). The resulting plasmid pGEM-T-P2P3 was digested with SpeI and PstI, and ligated to a XbaI/PstI DNA fragment prepared from pRN6441-gfp, which was made by cloning the green fluorescent protein (gfp) gene from pEBD166 (26Kane C.D. Schuch R. Day Jr., W.A. Maurelli A.T. J. Bacteriol. 2002; 184: 4409-4419Crossref PubMed Scopus (63) Google Scholar) (kindly provided by Dr. Kane at Uniformed Services University of the Health Sciences) plus the SD sequence from the sarA gene of S. aureus (27Cheung A.L. Projan S.J. J. Bacteriol. 1994; 176: 4168-4172Crossref PubMed Google Scholar). An EcoRI/PstI DNA fragment of pGEM-T-P2P3-gfp was then cloned into the EcoRI/PstI sites of pRN5543. Plasmid pWP1003 was constructed by cloning the XbaI/PstI DNA fragment of pRN6441-gfp into the XbaI/PstI sites of pRN5543. To construct pWP1103, a PCR product was prepared using oligonucleotides SINT3, 5′-GCGAATTCACATGAGAATTTTAGAAG-3′ (5′ of S. intermedius agrD (agrD-Si, GenBank accession number AY557375), EcoRI site, underlined) and SINT4, 5′-GCGATTCATGATTAATGATGATGATGATGATGTTTTTCCTCTTCTAACAACTCAGC-3′ (3′ end of agrD-Si; 6 histidine codons and a stop codon, italic; BspHI site, underlined) as primers and S. intermedius chromosomal DNA as a template. The EcoRI/BspHI-digested PCR product was then cloned into the EcoRI/BspHI sites of pLZ4012 (19Zhang L. Lin J. Ji G. J. Biol. Chem. 2004; 279: 19448-19456Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Plasmid pWP1104 was made by cloning a PCR product amplified from S. intermedius chromosomal DNA using primers SINT1 and SINT5 into the XbaI site of pWP1103. Plasmid pWP1104 (switch) was prepared as follows. PCR product A was generated using oligonucleotide GJ number 14, 5′-GCTCTAGATCGTATAATGACAG-3′ (before the SD sequence of the S. aureus group I agrB, XbaI site, underlined) and SINT24 5′-GGTGTTAATCACGACAACCTGCATCCCTAATCGTAC-3′ (amino acid residues 35–40 codons of S. intermedius agrB (agrB-Si), italic; amino acid residues 29–34 codons of S. aureus group I agrB, underlined) as primers and pRN6397 (the S. aureus group I agr cloned into E. coli vector pUC19 (3Novick R.P. Projan S.J. Kornblum J. Ross H.F. Ji G. Kreiswirth B. Vandenesch F. Moghazeh S. Mol. Gen. Genet. 1995; 248: 446-458Crossref PubMed Scopus (327) Google Scholar)) as a template. PCR product B was made by using oligonucleotides SINT23 (complementary to SINT24) and SINT5 as primers, and S. intermedius chromosomal DNA as a template. The PCR product A and B mixture (1:1) was used as the template for a PCR with primers GJ number 14 and SINT5. The final PCR product was then cloned into the XbaI site of pWP1103. Plasmid pWP1201 was constructed by cloning an NheI DNA fragment of pLZ4012 into the NheI site of the E. coli expression vector, pET11a. To make plasmid pWP1202, an PCR product using oligonucleotides GJ number 14 5′-GCTCTAGATCGTATAATGACAG-3′ (before the SD sequence of agrB, XbaI site, underlined) and GJ number 15-1, 5′-GCGTCTAGATCATTTTAAGTCCTCC-3′ (3′ of agrB, italic; XbaI site, underlined) as primers, and pRN6852 (5Ji G. Beavis R.C. Novick R.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 12055-12059Crossref PubMed Scopus (512) Google Scholar) as a template. After digestion with XbaI, the PCR product was cloned into the XbaI site of pWP1201. Site-directed mutagenesis of agrB-Si was done using PCR primers containing the desired mutations and the ExSite PCR-based site-directed mutagenesis method according to the manufacturer's instruction (Stratagene), resulting in plasmids pWP1111 to pWP1120 (Table I). Plasmid pWP1105 was constructed by cloning an XbaI DNA fragment containing the H77A mutation in agrB-Si prepared from pWP1118 into the XbaI site of pWP1102. The nucleotide sequences of the cloned wild type and mutated genes in the constructed plasmids were confirmed by DNA sequencing. Membrane Vesicle Preparation—S. aureus cells were grown in CYGP broth to 70 Klett units, and induced with 0.5 μg/ml methicillin at 37 °C for 3–5 h. Cells were harvested by centrifugation, washed with 1× sucrose-sodium maleate-MgCl2 (1 × SMM) (24Novick R.P. Methods Enzymol. 1991; 204: 587-636Crossref PubMed Scopus (468) Google Scholar), and suspended in 1× SMM containing 10 μg/ml lysostaphin. After 60 min incubation at 37 °C, protoplasts were prepared and washed with 1× SMM plus 5 mm EDTA. The protoplasts were then lysed by the addition of ice-cold buffer A (20 mm HEPES, pH 7.2, 5% glucose, 5 mm EDTA) followed by brief sonication. After centrifugation at 12,000 × g for 10 min at 4 °C to remove unlysed cells, the total cell lysates were centrifuged at 100,000 × g for 90 min at 4 °C to separate the cell membrane vesicles and the cytoplasmic fractions. The membrane vesicles were suspended in ice-cold buffer A for immediate use or snap-frozen in liquid nitrogen and stored at –80 °C until use. In Vitro Processing of AgrD—Membrane fusion experiments were performed using the freeze-thaw method as described (28Driessen A.J. Konings W.N. Methods Enzymol. 1993; 221: 394-408Crossref PubMed Scopus (27) Google Scholar). Equal volumes of membrane fractions prepared from S. aureus cells expressing AgrB or AgrD were mixed, and the mixtures were snap-frozen in liquid nitrogen followed by thawing at room temperature or at 4 °C three times to fuse the membranes. After incubation at 37 °C, the mixtures were centrifuged at 100,000 × g for 60 min at 4 °C. The supernatants were used for AIP activity assays, and the membrane pellets were suspended in 1× T7 bind/wash buffer (Novagen) plus 2% sodium cholate and protease inhibitor mixture set II (1:17 final dilution) (Calbiochem). The T7 epitope-tagged intact AgrD, processing intermediate(s), and final product(s) were purified by affinity chromatography using T7 monoclonal antibody agarose (Novagen) and suspended in 1× SDS-PAGE sample buffer (25Sambrook J. W. R.D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). Western Blot Hybridization—Samples were incubated at 42 °C for 30 min. Proteins were separated on Tris/Tricine SDS-PAGE (29Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10468) Google Scholar), and the separated proteins were then transferred to polyvinylidene difluoride membrane (Millipore). After incubation at 4 °C overnight, or at room temperature for 1 h in TBS buffer (25Sambrook J. W. R.D. Molecular Cloning: A Laboratory Manual. 3rd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar) plus 0.05% Tween 20 and 5% bovine serum albumin,
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