Article Figures & Data
Figures
- 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.
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.
- 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.
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.
- 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
- Table 1.
Primers and probes used for RT-PCR
Gene Forward primer Reverse primer Probe clfB TTCCAATGCGCAAGGAACTAG CAGCATTTACTACAGGTTCAGCAACT AGACTACGTACAGCTCTCGTTCTAACACTT gyrB CAAATGATCACAGCATTTGGTACAG CGGCATCAGTCATAATGACGAT AATCGGTGGCGACTTTGATCTAGCGAAAG hla AAAAAACTGCTAGTTATTAGAACGAAAGG GGCCAGGCTAAACCACTTTTG CCTTCTTCGCTATAAACTCTATATTGACCAGCAAT rnaIII GTGATGGAAAATAGTTGATGAGTTGTTT GAATTTGTTCACTGTGTCGATAATCC TGCACAAGATATCATTTCAACAATCAGTGACTTAGTAAAA sdrD TCAGATGAGCAAGCTTCACCAA TTGGTTGAGCATTTACCACTGATT ATTCTCTTGCAAATCAGGTTGTAACGCTTCTTG rot ATTTTGCAATTAGAAACACTTTTGG TCTTCTCTAGACATTTTGTATTCGCTTT TGACATTAACTCAATTTTCAGCGAGATTG sarH1 CCACCATAAATACCCTCAAACTGTT TCATCTTCAGTTGAGCGTTCTTTT AGCTCTCAATAATTTAAAAAAGCAAGGCTA sdrC CAACTGCAGATCAGCCTAAAGTGA TGGTGATTGCATGTTACTACTAGTTTCTT TGAGTGATAGTGCAACAGTT clfA AGGTTCTGGTGACGGTATCGA TCAATTTCACCAGGCTCATCAG AAACCAGTTGTTCCTGAAC efb TTTAACGATGGTACATTCGAATATGG ATCAGTTTTCGCTGCTGGTTTAT CACGTCCACAATTT mecA TTCCACATTGTTTCGGTCTAAAATT AATGCAGAAAGACCAAAGCATACA CCACGTTCTGATTTTAAA arcA1 TGCGATCGTATGTCACCACAA CAATGGAATGATGGCTCAAACA CCTGGTCGAATACATAAT arcA2 TCAGCTGCTAACTTCTCAAGGTAAAG CATTTTGCGCAGGTGCTAAG ACTTCAACACCCTCTTC spa CAGCAAACCATGCAGATGCTA GCTAATGATAATCCACCAAATACAGTTG CATTACCAGAAACTGGTGAAGAAAATCCATTCATTG - Table 2.
Selected agr-regulated genes in strains LAC, 252, and RN6390
Gene type and no. Gene Function Fold change in gene expression (wild-type/agr strain)a LAC 252 RN6390 Virulence SAUSA300_0113 spa Immunoglobulin G binding protein A 0.12 0.04 0.03 SAUSA300_0256 lrgA Murein hydrolase exporter NC 0.35 SAUSA300_0257 lrgB Murein hydrolase export regulator 0.09 SAUSA300_0320 geh Lipase (EC 3.1.1.3) 6.31 2.49 3.13 SAUSA300_0424 Low-affinity zinc transport protein 7.33 SAUSA300_0546 sdrC Neurexin binding protein 0.46 SAUSA300_0547 sdrD Surface binding protein 0.17 NP 0.06 SAUSA300_0548 sdrE Surface binding protein SAUSA300_0630 Multidrug resistance ABC transporter 7.51 10.26 5.70 SAUSA300_0772 clfA Clumping factor A (fibrinogen binding protein) 2.08 2.57 SAUSA300_0774 empbp Extracellular matrix binding protein/fibrinogen binding protein 0.36 SAUSA300_0776 nuc Thermonuclease (EC 3.1.31.1) 0.18 0.27 SAUSA300_0835 dltA d-Alanine-activating enzyme (EC 6.3.2.-) 0.33 0.46 SAUSA300_0836 dltB Protein DltB 0.33 0.33 SAUSA300_0837 dltC d-Alanyl carrier protein 0.43 0.41 SAUSA300_0838 dltD Protein DltD 0.27 0.39 0.49 SAUSA300_0949 sspC Hypothetical protein 6.79 27.33 SAUSA300_0950 sspB Staphopain (EC 3.4.22.-) 9.74 11.33 SAUSA300_0951 sspA Glutamyl endopeptidase (EC 3.4.21.19) 11.30 9.59 SAUSA300_1055 efb Fibrinogen binding protein 3.00 SAUSA300_1058 hla Alpha-hemolysin 2.17 0.25 23.68 SAUSA300_1067 psmβ1 Phenol-soluble modulin beta 1 173.35 61.61 41.55 SAUSA300_1068 psmβ2 Phenol-soluble modulin beta 2 63.24 NP 26.07 SAUSA300_1381 lukF-PV Leukocidin F subunit 7.14 NP NP SAUSA300_1382 lukS-PV Leukocidin S subunit 6.52 NP NP SAUSA300_1753 splF Serine protease (EC 3.4.21.-) 14.96 3.32 89.05 SAUSA300_1754 splE Serine protease (EC 3.4.21.-) 11.11 33.61 SAUSA300_1755 splD Serine protease (EC 3.4.21.-) 10.88 44.83 SAUSA300_1756 splC Serine protease (EC 3.4.21.-) 9.91 46.21 SAUSA300_1757 splB Serine protease (EC 3.4.21.-) 10.71 64.80 SAUSA300_1758 splA Serine protease (EC 3.4.21.-) 7.99 27.45 SAUSA300_1759 Hypothetical protein 69.53 NC 4.69 SAUSA300_1918 hlb Sphingomyelin phosphodiesterase (EC 3.1.4.12), truncated beta-toxin 6.39 8.85 SAUSA300_1922 sak Staphylokinase 0.49 SAUSA300_2440 fnbB Fibronectin binding protein 0.24 NP SAUSA300_2441 fnbA Fibronectin binding protein 2.87 SAUSA300_2603 lip Lipase (EC 3.1.1.3) 12.54 7.53 13.29 Resistance SAUSA300_0032 mecA MecA protein 4.70 NC SAUSA300_0033 mecR1 Methicillin resistance protein 3.02 NC SAUSA300_0928 comK Competence transcription factor 10.50 2.44 7.25 Metabolism SAUSA300_0061 arcC1 Carbamate kinase (EC 2.7.2.2) (ACME) NP NC SAUSA300_0062 arcB1 Ornithine carbamoyltransferase (EC 2.1.3.3) (ACME) 0.46 NP NC SAUSA300_0063 Transcription regulator, crp family (ACME) 0.45 NP NC SAUSA300_0064 arcD1 Arginine/ornithine antiporter (ACME) 0.40 NP NC SAUSA300_0065 arcA1 Arginine deiminase (EC 3.5.3.6) (ACME) 0.41 NP NC SAUSA300_0220 pflB Formate acetyltransferase (EC 2.3.1.54) 0.10 SAUSA300_0221 pflA Pyruvate formate-lyase-activating enzyme (EC 1.97.1.4) 0.19 SAUSA300_0311 Ribokinase (EC 2.7.1.15) 0.05 0.06 0.16 SAUSA300_0312 Sugar kinase 0.05 0.04 0.26 SAUSA300_0313 Nucleoside permease nupC 0.05 0.05 0.19 SAUSA300_0863 argH Argininosuccinate lyase (EC 4.3.2.1) 58.19 SAUSA300_0864 argG Argininosuccinate synthase (EC 6.3.4.5) 54.46 SAUSA300_1062 argF Ornithine carbamoyltransferase (EC 2.1.3.3) 0.07 0.12 0.31 SAUSA300_1063 arcC3 Carbamate kinase (EC 2.7.2.2) 0.08 0.19 0.30 SAUSA300_1712 ribH 6,7-Dimethyl-8-ribityllumazine synthase (EC 2.5.1.9) 31.50 SAUSA300_1713 ribBA GTP cyclohydrolase II (EC 3.5.4.25)/3,4-dihydroxy-2-butanone-4-phosphate synthase (EC 4.1.2.-) 15.58 SAUSA300_1714 ribE Riboflavin synthase alpha chain (EC 2.5.1.9) 42.18 SAUSA300_1715 ribD Diaminohydroxyphosphoribosylaminopyrimidine deaminase (EC 3.5.4.26)/5-amino-6-(5-phosphoribosylamino)uracil reductase (EC 1.1.1.193) 24.48 SAUSA300_2567 arcC2 Carbamate kinase (EC 2.7.2.2) 0.15 0.08 SAUSA300_2568 arcD2 Arginine/ornithine antiporter 0.15 0.21 SAUSA300_2569 arcB2 Ornithine carbamoyltransferase (EC 2.1.3.3) 0.18 0.11 SAUSA300_2570 arcA2 Arginine deiminase (EC 3.5.3.6) 0.22 0.05 Regulation SAUSA300_0605 sarA Staphylococcal accessory regulator 2.77 SAUSA300_0690 saeS Sensory transduction protein kinase (EC 2.7.3.-) 2.61 SAUSA300_0691 saeR Two-component response regulator 2.17 SAUSA300_1708 rot Staphylococcal accessory regulator 0.64 3.18 Transport SAUSA300_2453 ABC transporter ATP binding protein 0.05 0.09 0.20 SAUSA300_2454 ABC transporter ATP binding protein 0.06 0.06 0.15 ↵a NC, empty fields, no significant change; NP, gene not present in that strain.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Table S1. Microarray results.
MS Excel file, 419K.
- Supplemental file 1
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Table S1. Microarray results.
