Quorum Sensing in Staphylococci Is Regulated via Phosphorylation of Three Conserved Histidine Residues
2004; Elsevier BV; Volume: 279; Issue: 15 Linguagem: Inglês
10.1074/jbc.m311106200
ISSN1083-351X
AutoresYael Gov, Ilya Borovok, Moshe Korem, Vineet K. Singh, Radheshyam K. Jayaswal, Brian J. Wilkinson, Stephen M. Rich, Naomi Balaban,
Tópico(s)Biochemical and Structural Characterization
ResumoStaphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP– strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP– strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers. Staphylococcus aureus cause infections by producing toxins, a process regulated by cell-cell communication (quorum sensing) through the histidine-phosphorylation of the target of RNAIII-activating protein (TRAP). We show here that TRAP is highly conserved in staphylococci and contains three completely conserved histidine residues (His-66, His-79, His-154) that are phosphorylated and essential for its activity. This was tested by constructing a TRAP– strain with each of the conserved histidine residues changed to alanine by site-directed mutagenesis. All mutants were tested for pathogenesis in vitro (expression of RNAIII and hemolytic activity) and in vivo (murine cellulitis model). Results show that RNAIII is not expressed in the TRAP– strain, that it is non hemolytic, and that it does not cause disease in vivo. These pathogenic phenotypes could be rescued in the strain containing the recovered traP, confirming the importance of TRAP in S. aureus pathogenesis. The phosphorylation of TRAP mutated in any of the conserved histidine residues was significantly reduced, and mutants defective in any one of these residues were non-pathogenic in vitro or in vivo, whereas those mutated in a non-conserved histidine residue (His-124) were as pathogenic as the wild type. These results confirm the importance of the three conserved histidine residues in TRAP activity. The phosphorylation pattern, structure, and gene organization of TRAP deviates from signaling molecules known to date, suggesting that TRAP belongs to a novel class of signal transducers. Staphylococcus aureus are Gram-positive bacteria that are part of the normal flora but can become pathogenic and cause disease in humans and animals once they produce toxic exomolecules (1Lowy F.D. N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4673) Google Scholar). S. aureus pathogenesis is regulated by quorum sensing mechanisms (2Miller M.B. Bassler B.L. Annu. Rev. Microbiol. 2001; 55: 165-199Crossref PubMed Scopus (3429) Google Scholar, 3Sturme M.H. Kleerebezem M. Nakayama J. Akkermans A.D. Vaugha E.E. de Vos W.M. Antonie van Leeuwenhoek. 2002; 81: 233-243Crossref PubMed Scopus (222) Google Scholar, 4Kleerebezem M. Quadri L.E. Kuipers O.P. de Vos W.M. Mol. Microbiol. 1997; 24: 895-904Crossref PubMed Scopus (616) Google Scholar), where molecules (autoinducers) produced and secreted by the bacteria accumulate as a function of cell density. Once the autoinducers reach threshold concentration, they activate signal transduction pathways, leading to the expression of genes that code for virulence factors (1Lowy F.D. N. Engl. J. Med. 1998; 339: 520-532Crossref PubMed Scopus (4673) Google Scholar). To date two quorum-sensing systems have been described in S. aureus (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), which will be referred to herein as SQS 1 and SQS 2. SQS 1 consists of the autoinducer RNAIII-activating protein (RAP) 1The abbreviations used are: RAP, RNAIII-activating protein; TRAP, target of RAP; RIP, RNAIII-inhibiting peptide; agr, accessory gene regulator; WT, wild type. and its target molecule TRAP (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 6Balaban N. Goldkorn T. Nhan R.T. Dang L.B. Scott S. Ridgley R.M. Rasooly A. Wright S.C. Larrick J.W. Rasooly R. Carlson J.R. Science. 1998; 280: 438-440Crossref PubMed Scopus (199) Google Scholar). RAP is a 277-amino acid protein that is orthologous to the ribosomal protein L2 and is specifically secreted by S. aureus (7Korem M. Sheoran A.S. Gov Y. Tzipori S. Borovok I. Balaban N. FEMS Microbiol. Lett. 2003; 223: 167-175Crossref PubMed Scopus (51) Google Scholar). TRAP is a 167-amino acid protein that is constitutively expressed but is histidine-phosphorylated in a RAP-dependent manner (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and is, thus, suggested to act as the sensor of RAP. SQS 2 consists of the products of the accessory gene regulatory system agr. The chromosomal agrBDCA operon is active from the mid-exponential phase of growth and encodes two transcripts, RNAII and RNAIII (8Novick 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 (331) Google Scholar, 9Novick R.P. Ross H.F. Projan S.J. Kornblum J. Kreiswirth B. Moghazeh S. EMBO J. 1993; 12: 3967-3975Crossref PubMed Scopus (834) Google Scholar). RNAII encodes AgrB, AgrD, AgrC, and AgrA, where AgrD is a pro-peptide that yields a peptide pheromone (AIP) that is secreted with the aid of AgrB (10Lyon G.J. Wright J.S. Muir T.W. Novick R.P. Biochemistry. 2002; 41: 10095-100104Crossref PubMed Scopus (172) Google Scholar, 11Saenz H.L. Augsburger V. Vuong C. Jack R.W. Gotz F. Otto M. Arch. Microbiol. 2000; 174: 452-455Crossref PubMed Scopus (51) Google Scholar, 12Zhang L. Gray L. Novick R.P. Ji G. J. Biol. Chem. 2002; 277: 34736-34742Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). AgrC and AgrA are part of a bacterial two-component system, AgrC as the receptor component that is phosphorylated in an AIP ligand-dependent manner and AgrA as regulator (13Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (197) Google Scholar, 14Morfeldt E. Panova-Sapundjieva I Gustafsson B. Arvidson S. FEMS Microbiol. Lett. 1996; 143: 195-201Crossref PubMed Google Scholar). RNAIII acts as a regulatory RNA molecule to activate various toxin genes and also encodes for δ-hemolysin (15Arvidson S. Tegmark K. Int. J. Med. Microbiol. 2001; 291: 159-170Crossref PubMed Scopus (175) Google Scholar, 16Balaban N. Novick R.P. FEMS Microbiol. Lett. 1995; 133: 155-161PubMed Google Scholar, 17Benito Y. Kolb F.A. Romby P. Lina G. Etienne J. Vandenesch F. RNA (N. Y.). 2000; 6: 668-679Crossref PubMed Scopus (135) Google Scholar). The components of SQS1 and SQS2 interact with one another and the following scenario is, thus, emerging (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). As the cells multiply and the colony grows the cells secrete RAP. When RAP reaches a threshold concentration it induces the histidine phosphorylation of its target molecule TRAP, reaching peak phosphorylation at the mid-exponential phase of growth. The phosphorylation of TRAP leads, by a yet unknown mechanism, to activation of the agr and the production of RNAII (in the mid-exponential phase of growth) (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). AIP induces the phosphorylation of AgrC (13Lina G. Jarraud S. Ji G. Greenland T. Pedraza A. Etienne J. Novick R.P. Vandenesch F. Mol. Microbiol. 1998; 28: 655-662Crossref PubMed Scopus (197) Google Scholar), leading to the synthesis of RNAIII, which leads to the production of numerous secreted toxins (15Arvidson S. Tegmark K. Int. J. Med. Microbiol. 2001; 291: 159-170Crossref PubMed Scopus (175) Google Scholar). AIP also indirectly down-regulates the phosphorylation of TRAP (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). TRAP is hypothesized then, to be available for interacting with another RAP molecule, leading to a sustainable activation of agr and to the production of toxins (18Hong-Geller E. Gupta G. J. Mol. Recognit. 2003; 16: 91-101Crossref PubMed Scopus (21) Google Scholar). TRAP was first discovered as a quorum-sensing transducer of S. aureus (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and was then suggested to be a more global responder to stress, because it was overexpressed in cells exposed to antibiotic treatment (19Singh V.K. Jayaswal R.K. Wilkinson B.J. FEMS Microbiol. Lett. 2001; 199: 79-84PubMed Google Scholar). Like typical sensors of classical two component systems (4Kleerebezem M. Quadri L.E. Kuipers O.P. de Vos W.M. Mol. Microbiol. 1997; 24: 895-904Crossref PubMed Scopus (616) Google Scholar), TRAP is histidine-phosphorylated in the presence of RAP (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), and immunoelectron microscopy studies indicate that it is membrane-associated. 2N. Balaban, unpublished data. In addition, the C-terminal part of TRAP was used as a vaccine to protect from an infection, 3G. Yang, personal communication. suggesting that parts of the molecule may be extracellular. However, unlike classical sensors, TRAP does not contain a kinase or a transmembrane domain (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). It is, therefore, suggested that TRAP is a non-classical signal transducer and may be bound to the membrane through other proteins. The phosphorylation of TRAP has been suspected to be essential for S. aureus pathogenesis because in the presence of RIP, which is a linear heptapeptide that competes with RAP and inhibits TRAP phosphorylation, infections caused by staphylococci were prevented (5Balaban N. Goldkorn T. Gov Y. Hirshberg M. Koyfman N. Matthews H.R. Nhan R.T. Singh B. Uziel O. J. Biol. Chem. 2001; 276: 2658-2667Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 6Balaban N. Goldkorn T. Nhan R.T. Dang L.B. Scott S. Ridgley R.M. Rasooly A. Wright S.C. Larrick J.W. Rasooly R. Carlson J.R. Science. 1998; 280: 438-440Crossref PubMed Scopus (199) Google Scholar, 20Gov Y. Bitler A. Dell'Acqua G. Torres J.V. Balaban N. 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Circulation. 2003; 108: 767-771Crossref PubMed Scopus (64) Google Scholar, 26Balaban N. Giacometti A. Cirioni O. Gov Y. Ghiselli R. Mocchegiani F. Viticchi C. Del Prete M.S. Saba V. Scalise G. Dell'Acqua G. J. Infect. Dis. 2003; 187: 625-630Crossref PubMed Scopus (151) Google Scholar). Using mutagenesis and complementation studies we show here that TRAP is essential for S. aureus pathogenesis. Using site-directed mutagenesis studies we show here that the three conserved histidine residues are important for TRAP activity. Bacteria—S. aureus 8325-4 (further referred as WT); 8325-4 TRAP disrupted mutant strain (TRAP–) grown with 100 μg/ml kanamycin; S. aureus RN6390B (ATCC 55620); S. aureus NB8 (TRAP– strain containing recovered traP gene) grown with 100 μg/ml kanamycin and 10 μg/ml erythromycin, Staphylococcus epidermidis strain sofi, a clinical isolate of Tel Aviv University, Israel. Nares-isolates of S. aureus were obtained from patients at the onset of maintenance hemodialysis (21Balaban N. Gov Y. Bitler A. Boelaert J.R. Kidney Int. 2003; 63: 340-345Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Staphylococci were grown at 37 °C in tryptic soy broth, tryptic soy agar plates or on sheep blood agar plates (Remel, Lenexa, KS). Escherichia coli V2F1 (Invitrogen) and E. coli NS 2626 (Dam– strain) were grown in LB. DNA Manipulations—For E. coli, preparation of plasmids, DNA manipulations, and transformation of competent cells were as previously described (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For S. aureus genomic DNA was prepared as described (28Novick R.P. Methods Enzymol. 1991; 204: 587-636Crossref PubMed Scopus (473) Google Scholar). Standard procedures were employed for restriction enzyme digestion, ligation, Northern blotting, and radiolabeling of DNA probes (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) unless otherwise stated. The nucleotide sequences of the TRAP genomic regions in S. aureus clinical isolates were determined from both strands by the dideoxy procedure with the ABI Prism 377 automatic sequencer (PerkinElmer Life Sciences) and the ABI Prism dye terminator cycle sequencing kit (Applied Biosystems) and have been deposited in the GenBank™ data base with accession numbers AJ489447 (clinical isolate 12), AJ489448 (clinical isolate 15), AJ489449 (clinical isolate 11), and AJ489450 (clinical isolate 7). Amplification of traP by PCR—Forward (5′-GCGCGGATCCATCGCTCATTCGTTCG-3′) and reverse (5′-CGCGAAGCTTCCATTGGCATGTATGT-3′) primers were designed. Chromosomal DNA of S. aureus clinical nare isolates were used as templates to PCR-amplify the DNA fragment containing an intact traP gene as well as its putative promoter and transcriptional terminator. To amplify the traP gene in S. epidermidis, forward (5′-ATGTATTTATATACATCTTATGGGAC-3′) and reverse (5′-TTAATGATCTTCTATTGG-3′) primers were designed, and chromosomal DNA of S. epidermidis strain Sofi was used as a template. Sequence Analysis, Data Base Search, and Deduced Protein Analysis—Primary sequences of traP were identified in databases of the University of Oklahoma Advanced Center for Genome Technology (strain NCTC 8325) (www.genome.ou.edu/staph.html), The Institute for Genomic Research (strain COL) (www.tigr.org), and the S. aureus Sequencing Group at the Sanger Centre (EMRSA-16/MRSA252 and MSSA/strain 476) (www.sanger.ac.uk/Projects/S_aureus) using BLAST algorithms (BLASTn and tBLASTn) (29Altshul S.F. Madden T.L. Schaffer A.A. Zhang J Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) of databases including the NCBI Microbial Genomes Databases (www.ncbi.nlm.nih.gov/Microb_blast/unfinishedgenome.html). Multiple sequence alignments were made with the Sequencher™ program version 4.0 (GeneCodes). Phylogenetic comparison of the aligned nucleotide sequences was performed by the neighbor-joining method using the Tamura-Nei distance parameter in PAUP 4.0 (48Tamura K. Nei M. Mol. Biol. Evol. 1993; 10: 512-526PubMed Google Scholar, 49Nei M. Molecular Evolutionary Genetics. Columbia University Press, New York1987Crossref Google Scholar). Construction of TRAP-disrupted Mutant (TRAP–)—Two oligonucleotide primers P1 (upstream, 5′-GGATCCGACGCTAATGTAAATGGTG-3′) and P2 (downstream, 5′-AAGCTTTTCTATGTTGTACTCATTT-3′) were designed to PCR-amplify an ∼1.4-kilobase fragment encompassing the gene encoding for TRAP using S. aureus 8325-4 chromosomal DNA as template. The PCR product was cloned into pCR2.1 (Invitrogen). The cloned fragment was subsequently subcloned into the BamHI and HindIII sites of pTZ18RΔEcoRI. A kanamycin resistance cassette was subsequently inserted to the unique EcoRI site present in the traP gene. The vector pTZ18R cannot replicate in S. aureus, and the above construct was used as a suicide vector to transfer the mutation in S. aureus. Approximately 10 μg of pTZ-TRAP::kan DNA was electroporated to S. aureus RN4220 cells, and the transformants were selected on tryptic soy agar plates containing 100 μg/ml kanamycin. Southern blot and PCR analysis revealed that the resulting transformants possessed both the native and the mutated gene, suggesting that the entire construct was integrated into the chromosome due to a single crossover event. The mutation in the gene-encoding TRAP was subsequently transduced by preparing phage 80α lysates of the above transformants and infecting the S. aureus 8325-4 with that lysate. The transductants with mutations in the traP gene were subsequently confirmed by PCR and Southern blot analysis. Restoring TRAP Activity—Primers P1 (upstream, 5′-GGATCCGACGCTAATGTAAATGGTG-3′) and P2 (downstream, 5′-AAGCTTTTCTATGTTGTACTCATTT-3′) were designed to PCR-amplify an 888-kilobase fragment encompassing the gene encoding for TRAP, including its promoter region and termination site and BamHI/HindIII digestion sites (underlined), using S. aureus RN6390 chromosomal DNA as template. The PCR product ("whole traP") was digested and cloned into the BamHI/HindIII sites of pAUL-A, which can replicate in E. coli at 37 °C and in S. aureus at 30 °C and can be used as a suicide vector if grown at 37 °C in S. aureus. pAUL-A contains the gene for erythromycin resistance. The resulting plasmid (pAUL-A::traP (pYG14) was used to transform (by electroporation) E. coli V2F1. Cells were selected on LB agar plates containing 300 μg/ml erythromycin. Plasmid was isolated from positive clones and used to subclone (by electroporation) E. coli NS 2626 Dam– strain, and positive clones were selected on LB agar plates with 300 μg/ml erythromycin and 25 μg/ml chloramphenicol. The plasmid was isolated from positive clones and used to transform S. aureus RN4220 cells, and transformants were selected on tryptic soy agar plates containing 10 μg/ml erythromycin at 30 °C. A positive clone (RN4220 containing pYG14 (NB6)) was selected, the plasmid was isolated, and the presence of pYG14 was confirmed by PCR (using forward M13 primer and reverse 3′ TRAP primer) followed by DNA sequencing. For transduction phage ϕ11 was used to produce a phage lysate of strain NB6. The lysate was then used to infect the recipient strain S. aureus TRAP–, and infected cells were selected by growing them for 24–48 h at 37 °C with 10 μg/ml erythromycin and 100 μg/ml kanamycin. Colonies that grew were tested for the presence pYG14 by PCR (using forward and reverse traP primers), and positive clone (NB8) was grown on sheep blood agar plate and tested positively for hemolytic activity. Site-directed Mutagenesis Studies—In-frame mutations of residues His-66, His-79, His-154, and His-124 were introduced within the traP-coding region, where plasmid pYG14 (pAUL-A::traP) was used as a template for mutagenesis. Two complimentary oligonucleotide pairs were designed for each mutagenesis (primers 3 and 4 for His-66, primers 5 and 6 for His-79, primers 7 and 8 for His-154, and primers 9 and 10 for His-124 (Table I). Primers 1 and 2 were used to amplify the whole gene containing the mutation (Table I). To introduce a mutation, for example, in His-66 and replace it with Ala (H66A), two PCR reactions were carried out using the 1 + 4 and 2 + 3 primers and the S. aureus 8325-4 DNA as a template. The PCR products were purified (Roche Applied Science kit) and treated with Klenow to remove a hanging A nucleotide, and DNA fragments containing the mutations were then gel-purified. Purified PCR products were mixed and used as templates to amplify the whole gene containing the mutation using external primers 1 + 2. The PCR product (whole traP containing Ala-66 instead of His-66) was digested and cloned into the BamHI/HindIII sites of pAUL-A. The resulting plasmid (whole Ala66 traP::pAUL-A (pYG7)) was used to transform (by electroporation) E. coli V2F1. Cells were selected on LB agar plates containing 300 μg/ml erythromycin. The plasmid was isolated from positive clones. The in-frame mutation within traP in pAUL-A was confirmed by DNA sequencing, the plasmid was used to subclone (by electroporation) E. coli NS 2626 Dam– strain, and positive clones were selected on LB agar plates with 300 μg/ml erythromycin and 25 μg/ml chloramphenicol. The plasmid was isolated from positive clones and used to transform S. aureus RN4220 cells, and transformants were selected on tryptic soy agar plates containing 10 μg/ml erythromycin at 30 °C. A positive clone (YG4) was selected, the plasmid was isolated, and the presence of the mutation was confirmed by PCR (using forward M13 primer and reverse 3′ TRAP primer) followed by DNA sequencing. For transduction, phage ϕ11 was used to produce a phage lysate of strain YG4. The lysate was then used to infect the recipient strain S. aureus TRAP–, and infected cells were selected by growing them for 24–48 h at 37 °C with 10 μg/ml erythromycin and 100 μg/ml kanamycin. Colonies were tested for the presence of traP by PCR, and positive clone were sequenced to ensure the presence of the mutation.Table IPrimers used to prepare H66A, H79A, H154A, and H124A) (CAT to GCT)5′-GGATCCGACGCTAATGTAAATGGTG-3′ (upstream of traP promoter region, containing the BamHI site (underlined)5′-AAGCTTTTCTATGTTGTACTCATT-3′ (downstream of traP termination site; the HindIII site is underlined)5′-TTCAGTGAACATGCTTTCTATTGTGCAATC-3′ (His-66 mutated to Ala (bold), forward)5′-TGCACAATAGAAAGCATGTTCACTGAATTC-3′ (His-66 mutated to Ala (bold), reverse)5′-TCAACAGAAGATGCTGCATATCAACTTGAA-3′ (His-79 mutated to Ala (bold), forward)5′-AAGTTGATATGCAGCATCTTCTGTTGATGG-3′ (His-79 mutated to Ala (bold), reverse)5′-CAAGCGGACAAGCTTCAAGTTATTTTGAAA-3′ (His-154 mutated to Ala (bold), forward)5′-AAATAACTTGAAGCTTGTCCGCTTGAACCA-3′ (His-154 mutated to Ala (bold), reverse)5′-TTTGCTGATCGAGCTGCATACGAAGACTTT-3′ (His-124 mutated to Ala (bold), forward)5′-GTCTTCGTATGCAGCTCGATCAGCAAATAA-3′ (His-124 mutated to Ala (bold), reverse) Open table in a new tab In Vivo Phosphorylation—Cells (5 ml) were grown to the early exponential phase of growth, collected by centrifugation, and resuspended in 0.9 ml of low phosphate buffer (20 mm KCl, 80 mm NaCl, 20 mm NH4Cl, 0.14 mm Na2SO4, 100 mm Tris, pH 7.4, 2.5 mm MgCl2, 0.1 mm CaCl2, 2 mm FeCl2, 0.4% glucose, 9 mg/ml thiamine, 0.8 mm potassium phosphate buffer, pH 7.4, 0.25 mm l-arginine, 0.21 mm l-histidine, 0.62 mm l-lysine, 0.13 mm l-glutamic acid, 0.056 mm glycine, 0.32 mm l-alanine, 0.46 mm l-valine, 0.36 mm l-isoleucine, 0.82 mm l-proline, 0.016 mm l-phenylalanine, 0.50 mm l-serine, 0.34 mm l-threonine, 0.016 mm l-tyrosine, 0.27 mm l-cysteine, 0.21 mm l-methionine, 0.13 mm l-asparagine, 0.029 mm nicotinic acid, 0.13 mm l-glutamine) and 28 mCi of radiolabeled orthophosphate (32P) (ICN Biochemicals). Cells were grown with shaking for 40 min at 37 °C for the times indicated. Cells were collected by centrifugation and washed once in phosphate-buffered saline, and cells were resuspended in 20 μl of 50 mg/ml lysostaphin in 10 mm Tris, pH 8.0, 1 mm EDTA for 10 min at room temperature, Laemmli sample buffer was added (without boiling), and the sample (total cell homogenate) was separated by 15% SDS-PAGE. The gel was autoradiographed and then stained in Coomassie to ensure that equal amounts of protein were in fact loaded on the gel. Detection of RNAIII; RNA Purification and Northern Blot Analysis— Early exponential cells were grown from A600 0.03 for several hours with shaking at 37 °C. At timed intervals cells (∼3 × 108) were collected by centrifugation (2 min, 12,000 × g) and resuspended in 20 μl of lysostaphin in TES buffer (100 μg/ml lysostaphin (Sigma-Aldrich) in 100 mm Tris, pH 7.2, 1 mm EDTA, 20% sucrose) and incubated for 10 min at room temperature. 20 μl of 2% SDS containing proteinase K (100 μg/ml) was added and vigorously vortexed for 1 min followed by 10 min of incubation at room temperature. The sample was frozen and thawed twice. 15 μl of RNA sample (∼1 × 108 cells) was mixed with 11% deionized glyoxal, 16 mm phosphate buffer, pH 7.0, and 55% Me2SO (final concentrations) and incubated for 1 h at 65 °C. RNA loading buffer (Ambion) was added, and sample was applied to a 1% agarose gel in 10 mm phosphate buffer, pH 7.0, supplemented with 5 mm iodoacetic acid (Sigma-Aldrich). Gel was Northern-blotted by dry transfer. The membrane was prehybridized using rapid-hyb (Amersham Biosciences) followed by hybridization with PCR-radiolabeled RNAIII-specific DNA (amplified by PCR using the forward primer, 5′-ATGATCACAGAGATGTGA-3′, and reverse primer, 5′-CTGAGTCCTAGGAAACTAACT-3′), using S. aureus RN6390B chromosomal DNA as a template. Membranes were autoradiographed. To test for RNAIII in all mutant strains cells were grown from the early exponential phase of growth for several hours (to A600 = 3.0) and analyzed for RNAIII as above. Cellulitis Mouse Model—Bacteria were grown overnight at 37 °C on tryptic soy agar plates and collected into sterile saline. Bacteria (1 × 109 colony-forming units in 100 μl saline) were injected subcutaneously together with 1 mg of sterile Cytodex beads (Sigma) into 7-week-old immunocompetent hairless mice (Crl:SKH1-hrBR, Charles River Laboratories) (n = 8). Animals were observed daily for mortality, overall health, and development of lesions. The size of the lesions were measured several days post-challenge (area = 0.5 (π(length)(width))). Statistical Analysis—Statistical analysis was performed using Student's t test by Microsoft Excel (Microsoft). Significance was accepted when the p value was ≤0.05. TRAP Is Unique to Staphylococci but Is Conserved among Strains and Species—The traP gene in various clinical isolates of S. aureus and S. epidermidis was amplified by PCR, and its sequence was determined. Multiple sequence alignment of traP from multiple strains of both species indicates that the traP gene sequence is highly conserved. However, individual traP nucleotide and deduced amino acid sequences can be divided into subgroups (Fig. 1, A and B). Our analysis of nucleotide sequences of the strains in the present study are consistent with those of Gilot et al. (34Gilot P. Lina G Cochard T. Poutrel B. J. Clin. Microbiol. 2002; 40: 4060-4067Crossref PubMed Scopus (219) Google Scholar), who previously grouped mastitis agents into four major groups based on MseI restriction fragment length polymorphisms. These authors reported a grouping designated as type I, which includes NCTC 8325, COL (33Shafer W.M. Iandolo J.J. Infect. Immun. 1979; 25: 909-911Crossref Google Scholar), Mu50 (VRSA), and N315 (MRSA) (31Kuroda M. Ohta T. Uchiyama I. Baba T. Yuzawa H. Kobayashi I. Cui L. Oguchi A. Aoki K. Nagai Y. Lian J. Ito T. Kanamori M. Matsumaru H. Maruyama A. Murakami H. Hosoyama A. Mizutani-Ui Y. Takahashi N.K. Sawano T. Inoue R. Kaito C. Sekimizu K. Hirakawa H. Kuhara S. Goto S. Yabuzaki J. Kanehisa M. Yamashita A. Oshima K. Furuya K. Yoshino C. Shiba T. Hattori M. Ogasawara N. Hayashi H. Hiramatsu K. Lancet. 2001; 357: 1225-1240Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar). Type II and type III consists of strains EMRSA-16/MRSA252 and MSSA476, respectively; a fourth group was evident and is represented in the current set as mastitis type IV. In the present study we found that clinical nare isolates 7 and 11 corresponded to type I, whereas clinical nare isolates 12 and 15 were quite similar to strain WHO260001. The latter set of three isolates comprises a fifth subgroup (type V), which was not reported by Gilot et al. (34Gilot P. Lina G Cochard T. Poutrel B. J. Clin. Microbiol. 2002; 40: 4060-4067Crossref PubMed Scopus (219)
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