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Molecular Pathogenesis

Role of the Accessory Gene Regulator agr in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis

Gordon Y. C. Cheung, Rong Wang, Burhan A. Khan, Daniel E. Sturdevant, Michael Otto
S. M. Payne, Editor
Gordon Y. C. Cheung
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, Maryland 20892
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Rong Wang
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, Maryland 20892
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Burhan A. Khan
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, Maryland 20892
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Daniel E. Sturdevant
2Research Technologies Branch, Genomics Unit, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Hamilton, Montana 59840
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Michael Otto
1Laboratory of Human Bacterial Pathogenesis, National Institute of Allergy and Infectious Diseases, The National Institutes of Health, Bethesda, Maryland 20892
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  • For correspondence: motto@niaid.nih.gov
S. M. Payne
Roles: Editor
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DOI: 10.1128/IAI.00046-11
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Figures

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  • Fig. 1.
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    Fig. 1.

    Effect of agr on virulence in a mouse subcutaneous infection model. (A) Abscess sizes. Bacteria of the indicated strains were injected subcutaneously in hairless mice at ∼3 × 107 CFU (except RN6390 and RN6911, 1 × 106 CFU), and abscess dimensions were measured every day. Statistical significance of differences between abscess sizes on each day for each of the three wild-type/agr mutant comparisons was determined using unpaired t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; #, all values achieved with the agr mutant strain were 0. (B) Development of dermonecrotic lesions. Mice infected with the LAC and RN6390 strains, but not the other strains, commonly developed open dermonecrotic lesions as shown in the upper panel. The lower panel shows a characteristic closed abscess formed by the LAC agr strain. (C) Histopathological evaluation of abscesses formed by the LAC and LAC agr strains. Histopathology of abscesses formed by RN6390 was similar to that of abscesses formed by strain LAC, and histopathologies of abscesses formed by the 252, 252 agr, and RN6911 strains were similar to those of abscesses formed by the LAC agr strain.

  • Fig. 2.
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    Fig. 2.

    Growth-dependent expression of agr. Expression of agr was determined using qRT-PCR of RNAIII during the growth of S. aureus strains and their isogenic agr mutants. Lines represent OD600, plotted on the left y axis. Bars represent the relative expression of RNAIII compared to that of the housekeeping gene gyrB (control), plotted on the right y axis. Expression of RNAIII in agr-negative strains was not detectable in any strain at any time point.

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    Fig. 3.

    Effect of agr on PSM expression in various strain backgrounds. (A) Expression of the psmα and psmβ operons was determined using qRT-PCR as previously described (35). (B) Concentrations of all S. aureus PSMs (see legend) in culture filtrates were measured as described previously (35). Samples for measurements were taken from 8-h (strains LAC and RN6390) or 10-h (strain 252) cultures. Striped parts of bars represent the N-deformylated fraction of a particular PSM. Note that strain 252 contains the psm-mec gene in contrast to strains LAC and RN6390.

  • Fig. 4.
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    Fig. 4.

    Effect of agr on the expression of selected surface protein and regulatory genes in various strain backgrounds. The graphs show expression of selected genes as determined by qRT-PCR at maximal expression of agr in the LAC, LAC agr, RN6390, and RN6911 strains for 8 h and the 252 and 252 agr strains for 10 h. Data shown on the y axis represent expression relative to that of the housekeeping gene gyrB (control). *, P < 0.05; **, P < 0.01; ***, P < 0.001 (t tests comparing agr-negative to wild-type strain samples). Expression levels of alpha-toxin were confirmed on the protein level by Western blots with specific alpha-toxin antiserum and are shown above the qRT-PCR data.

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    Fig. 5.

    Effect of agr on methicillin resistance and fibrinogen binding phenotypes. (A) MIC assays with oxacillin. Strains were inoculated from precultures and grown with the addition of oxacillin at different concentrations in microtiter plates for 24 h. (B) Fibrinogen binding capacity. Bacteria were grown for 16 h and assayed for fibrinogen binding capacity on fibrinogen-coated microtiter plates after an incubation period of 2 h. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Tables

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  • Additional Files
  • Table 1.

    Primers and probes used for RT-PCR

    GeneForward primerReverse primerProbe
    clfBTTCCAATGCGCAAGGAACTAGCAGCATTTACTACAGGTTCAGCAACTAGACTACGTACAGCTCTCGTTCTAACACTT
    gyrBCAAATGATCACAGCATTTGGTACAGCGGCATCAGTCATAATGACGATAATCGGTGGCGACTTTGATCTAGCGAAAG
    hlaAAAAAACTGCTAGTTATTAGAACGAAAGGGGCCAGGCTAAACCACTTTTGCCTTCTTCGCTATAAACTCTATATTGACCAGCAAT
    rnaIIIGTGATGGAAAATAGTTGATGAGTTGTTTGAATTTGTTCACTGTGTCGATAATCCTGCACAAGATATCATTTCAACAATCAGTGACTTAGTAAAA
    sdrDTCAGATGAGCAAGCTTCACCAATTGGTTGAGCATTTACCACTGATTATTCTCTTGCAAATCAGGTTGTAACGCTTCTTG
    rotATTTTGCAATTAGAAACACTTTTGGTCTTCTCTAGACATTTTGTATTCGCTTTTGACATTAACTCAATTTTCAGCGAGATTG
    sarH1CCACCATAAATACCCTCAAACTGTTTCATCTTCAGTTGAGCGTTCTTTTAGCTCTCAATAATTTAAAAAAGCAAGGCTA
    sdrCCAACTGCAGATCAGCCTAAAGTGATGGTGATTGCATGTTACTACTAGTTTCTTTGAGTGATAGTGCAACAGTT
    clfAAGGTTCTGGTGACGGTATCGATCAATTTCACCAGGCTCATCAGAAACCAGTTGTTCCTGAAC
    efbTTTAACGATGGTACATTCGAATATGGATCAGTTTTCGCTGCTGGTTTATCACGTCCACAATTT
    mecATTCCACATTGTTTCGGTCTAAAATTAATGCAGAAAGACCAAAGCATACACCACGTTCTGATTTTAAA
    arcA1TGCGATCGTATGTCACCACAACAATGGAATGATGGCTCAAACACCTGGTCGAATACATAAT
    arcA2TCAGCTGCTAACTTCTCAAGGTAAAGCATTTTGCGCAGGTGCTAAGACTTCAACACCCTCTTC
    spaCAGCAAACCATGCAGATGCTAGCTAATGATAATCCACCAAATACAGTTGCATTACCAGAAACTGGTGAAGAAAATCCATTCATTG
  • Table 2.

    Selected agr-regulated genes in strains LAC, 252, and RN6390

    Gene type and no.GeneFunctionFold change in gene expression (wild-type/agr strain)a
    LAC252RN6390
    Virulence
        SAUSA300_0113spaImmunoglobulin G binding protein A0.120.040.03
        SAUSA300_0256lrgAMurein hydrolase exporterNC0.35
        SAUSA300_0257lrgBMurein hydrolase export regulator0.09
        SAUSA300_0320gehLipase (EC 3.1.1.3)6.312.493.13
        SAUSA300_0424Low-affinity zinc transport protein7.33
        SAUSA300_0546sdrCNeurexin binding protein0.46
        SAUSA300_0547sdrDSurface binding protein0.17NP0.06
        SAUSA300_0548sdrESurface binding protein
        SAUSA300_0630Multidrug resistance ABC transporter7.5110.265.70
        SAUSA300_0772clfAClumping factor A (fibrinogen binding protein)2.082.57
        SAUSA300_0774empbpExtracellular matrix binding protein/fibrinogen binding protein0.36
        SAUSA300_0776nucThermonuclease (EC 3.1.31.1)0.180.27
        SAUSA300_0835dltAd-Alanine-activating enzyme (EC 6.3.2.-)0.330.46
        SAUSA300_0836dltBProtein DltB0.330.33
        SAUSA300_0837dltCd-Alanyl carrier protein0.430.41
        SAUSA300_0838dltDProtein DltD0.270.390.49
        SAUSA300_0949sspCHypothetical protein6.7927.33
        SAUSA300_0950sspBStaphopain (EC 3.4.22.-)9.7411.33
        SAUSA300_0951sspAGlutamyl endopeptidase (EC 3.4.21.19)11.309.59
        SAUSA300_1055efbFibrinogen binding protein3.00
        SAUSA300_1058hlaAlpha-hemolysin2.170.2523.68
        SAUSA300_1067psmβ1Phenol-soluble modulin beta 1173.3561.6141.55
        SAUSA300_1068psmβ2Phenol-soluble modulin beta 263.24NP26.07
        SAUSA300_1381lukF-PVLeukocidin F subunit7.14NPNP
        SAUSA300_1382lukS-PVLeukocidin S subunit6.52NPNP
        SAUSA300_1753splFSerine protease (EC 3.4.21.-)14.963.3289.05
        SAUSA300_1754splESerine protease (EC 3.4.21.-)11.1133.61
        SAUSA300_1755splDSerine protease (EC 3.4.21.-)10.8844.83
        SAUSA300_1756splCSerine protease (EC 3.4.21.-)9.9146.21
        SAUSA300_1757splBSerine protease (EC 3.4.21.-)10.7164.80
        SAUSA300_1758splASerine protease (EC 3.4.21.-)7.9927.45
        SAUSA300_1759Hypothetical protein69.53NC4.69
        SAUSA300_1918hlbSphingomyelin phosphodiesterase (EC 3.1.4.12), truncated beta-toxin6.398.85
        SAUSA300_1922sakStaphylokinase0.49
        SAUSA300_2440fnbBFibronectin binding protein0.24NP
        SAUSA300_2441fnbAFibronectin binding protein2.87
        SAUSA300_2603lipLipase (EC 3.1.1.3)12.547.5313.29
    Resistance
        SAUSA300_0032mecAMecA protein4.70NC
        SAUSA300_0033mecR1Methicillin resistance protein3.02NC
        SAUSA300_0928comKCompetence transcription factor10.502.447.25
    Metabolism
        SAUSA300_0061arcC1Carbamate kinase (EC 2.7.2.2) (ACME)NPNC
        SAUSA300_0062arcB1Ornithine carbamoyltransferase (EC 2.1.3.3) (ACME)0.46NPNC
        SAUSA300_0063Transcription regulator, crp family (ACME)0.45NPNC
        SAUSA300_0064arcD1Arginine/ornithine antiporter (ACME)0.40NPNC
        SAUSA300_0065arcA1Arginine deiminase (EC 3.5.3.6) (ACME)0.41NPNC
        SAUSA300_0220pflBFormate acetyltransferase (EC 2.3.1.54)0.10
        SAUSA300_0221pflAPyruvate formate-lyase-activating enzyme (EC 1.97.1.4)0.19
        SAUSA300_0311Ribokinase (EC 2.7.1.15)0.050.060.16
        SAUSA300_0312Sugar kinase0.050.040.26
        SAUSA300_0313Nucleoside permease nupC0.050.050.19
        SAUSA300_0863argHArgininosuccinate lyase (EC 4.3.2.1)58.19
        SAUSA300_0864argGArgininosuccinate synthase (EC 6.3.4.5)54.46
        SAUSA300_1062argFOrnithine carbamoyltransferase (EC 2.1.3.3)0.070.120.31
        SAUSA300_1063arcC3Carbamate kinase (EC 2.7.2.2)0.080.190.30
        SAUSA300_1712ribH6,7-Dimethyl-8-ribityllumazine synthase (EC 2.5.1.9)31.50
        SAUSA300_1713ribBAGTP cyclohydrolase II (EC 3.5.4.25)/3,4-dihydroxy-2-butanone-4-phosphate synthase (EC 4.1.2.-)15.58
        SAUSA300_1714ribERiboflavin synthase alpha chain (EC 2.5.1.9)42.18
        SAUSA300_1715ribDDiaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26)/5-amino-6-(5-phosphoribosylamino)uracil reductase (EC 1.1.1.193)24.48
        SAUSA300_2567arcC2Carbamate kinase (EC 2.7.2.2)0.150.08
        SAUSA300_2568arcD2Arginine/ornithine antiporter0.150.21
        SAUSA300_2569arcB2Ornithine carbamoyltransferase (EC 2.1.3.3)0.180.11
        SAUSA300_2570arcA2Arginine deiminase (EC 3.5.3.6)0.220.05
    Regulation
        SAUSA300_0605sarAStaphylococcal accessory regulator2.77
        SAUSA300_0690saeSSensory transduction protein kinase (EC 2.7.3.-)2.61
        SAUSA300_0691saeRTwo-component response regulator2.17
        SAUSA300_1708rotStaphylococcal accessory regulator0.643.18
    Transport
        SAUSA300_2453ABC transporter ATP binding protein0.050.090.20
        SAUSA300_2454ABC transporter ATP binding protein0.060.060.15
    • ↵a NC, empty fields, no significant change; NP, gene not present in that strain.

Additional Files

  • Figures
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  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Table S1. Microarray results.
      MS Excel file, 419K.
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Role of the Accessory Gene Regulator agr in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis
Gordon Y. C. Cheung, Rong Wang, Burhan A. Khan, Daniel E. Sturdevant, Michael Otto
Infection and Immunity Apr 2011, 79 (5) 1927-1935; DOI: 10.1128/IAI.00046-11

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Role of the Accessory Gene Regulator agr in Community-Associated Methicillin-Resistant Staphylococcus aureus Pathogenesis
Gordon Y. C. Cheung, Rong Wang, Burhan A. Khan, Daniel E. Sturdevant, Michael Otto
Infection and Immunity Apr 2011, 79 (5) 1927-1935; DOI: 10.1128/IAI.00046-11
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