Article Figures & Data
Figures
- FIG 1
PI-PLC activity and expression vary among S. aureus strains. (A) Kinetic assay demonstrating the PI-PLC activity associated with various S. aureus culture supernatants using a fluorescent artificial substrate over a 30-min time course. The results represent the means for three independent experiments. (B) PI-PLC activity normalized to the protein concentration for culture supernatants. Results are the ratio between the maximum relative fluorescent units (RFU) and protein concentration for a given strain and represent the means ± SEMs for three independent experiments. (C) Representative immunoblot (IB) visualizing the PI-PLC present in the culture supernatants using a monoclonal antibody against PI-PLC. Immunoreactivity was quantitated on a phosphorimager, and the percentages of immunoreactive protein were quantitated for all strains using the TCH1516 signal, which was set to 100%. Abbreviations: New, S. aureus Newman; Col, S. aureus COL; LAC, S. aureus USA300 LAC; TCH, S. aureus USA300 TCH1516; MW2, S. aureus USA400 MW2; 252, S. aureus USA200 MRSA252; UAM, S. aureus USA200 UAMS-1; 600, S. aureus USA600 NRS22.
- FIG 2
Contribution of S. aureus TCSs to the regulation and expression of plc. (A) S. aureus wild-type and TCS mutant strains in USA300 were grown in TSB to late-log growth phase (OD550 = 1.5). The relative expression of plc in each strain was compared. The expression level of plc in each strain was normalized to that of the DNA gyrase subunit B (gyrB). (B) Normalized PI-PLC activity data obtained for each of the TCS mutants from overnight supernatants in relation to those for the wild type. These results represent the means ± SEMs from three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01.
- FIG 3
Characterization of the plc promoter region. (A) Depiction of the upstream region of plc. Key features include the annotated translational start site (plc), the identified transcriptional start site (+1), a predicted ribosomal binding site (RBS), and a predicted −10 promoter element (−10). (B) The region directly upstream of plc was bound by phosphorylated His-SrrA. Approximately 5 ng of probe was used for all reactions. Reaction lanes include no His-SrrA (lane 1), various amounts of His-SrrA (lanes 2 to 6), nonfluorescent specific competitor DNA (lane 7), and nonfluorescent nonspecific competitor DNA (lane 8). The concentration of His-SrrA added to each reaction lane is listed. Specific and nonspecific competitor DNA was added at a roughly 50-fold molar excess. Data are representative of those from two independent experiments.
- FIG 4
Regulation of S. aureus plc and srrA in response to in vitro and in vivo oxidative stress. The expression of plc (A) or srrA (B) was compared to that in PBS-treated controls by qRT-PCR in vitro, using various concentrations of either HOCl or H2O2 as sources of oxidative stress for 60 min. In addition, levels of plc (C) or srrA (D) expression were examined in PMNs 10 min after phagocytosis. PMNs treated with DPI or obtained from a patient who suffers from CGD were compared with normal PMNs. Controls included bacteria grown in broth, bacteria that had undergone 20 min of opsonization (Ops), and bacteria that had been resuspended in the PMN buffer for 10 min (Buf). For statistical analysis, all comparisons are against the broth-grown controls. The levels of plc and srrA expression were normalized to the level of expression of the DNA gyrase subunit B (gyrB). All results represent the means ± SEMs from three independent experiments. *, P < 0.05; **, P < 0.01.
- FIG 5
Contribution of SrrAB and AgrAC TCSs to plc regulation. The relative expression of plc was examined in an S. aureus USA300 ΔsrrAB background (A) or a Δagr background (C) after 60 min of exposure to PBS or various concentrations of HOCl. In addition, the relative expression of plc in PMNs at 10 min postphagocytosis was examined in an S. aureus USA300 ΔsrrAB background (B) or Δagr background (D). Controls included broth-grown bacteria and bacteria that have been opsonized for 20 min (Ops). The level of plc expression was normalized to that of the DNA gyrase subunit B (gyrB), and results represent the means ± SEMs from three independent experiments. **, P < 0.01.
- FIG 6
Construction of the Δplc mutant in S. aureus. (A) Chromosomal organization of plc and surrounding genes in an S. aureus USA300 background. The primer sets used for determining genomic deletion are shown. (B) Gel depicting the PCRs run for potential Δplc mutants using the primer set depicted in panel A. Lanes: L, ladder; WT, S. aureus USA300 wild-type genomic DNA; Δ, S. aureus USA300 Δplc genomic DNA. (C) Immunoblot of S. aureus derivative supernatants using goat polyclonal antibody against PI-PLC. Lanes: WT, wild type; Δ, Δplc mutant; C, complemented strain. (D) PI-PLC activity for S. aureus derivatives determined by PI-PLC assay for 30 min. Results for panel D represent the means from three independent experiments performed in triplicate.
- FIG 7
Viability of S. aureus derivatives in whole-blood assays. The viability of S. aureus USA300 Δplc (A and B; stationary phase), ΔsrrAB (C; exponential phase), or ΔsrrAB (D; exponential phase) strains overexpressing PI-PLC exposed to human whole blood was calculated as the number of CFU recovered after 3 h of exposure divided by the number of CFU in the initial bacterial challenge. The numbers of CFU were determined by serial dilution and plating on TSA plates. One set of experiments (A) compared the viability in human blood of the wild-type strain and the Δplc mutant suspended in their own conditioned media or washed (w) and suspended in sterile culture medium. The strains used in experiments involving complementation harbored either control plasmids or the complementing plasmid. The results represent the means ± SEMs for three independent experiments. *, P < 0.05.
- FIG 8
Survival of S. aureus Δplc strains in PMNs. PMNs were challenged with wild-type or Δplc mutant S. aureus USA300 strains grown to mid-log phase at an MOI of either 1:1 (A) or 5:1 (B). Survival was determined by measuring the mean fluorescence index (MFI) of PMNs that had been challenged with GFP-expressing S. aureus derivatives. The results represent the means ± SEMs from at least three independent experiments. Times are in minutes (m). *, P < 0.05.
- FIG 9
Challenge of mice with S. aureus USA300 Δplc strains. (A) BALB/c mice were challenged with a bolus of 5 × 106 CFU of wild-type or Δplc mutant S. aureus by tail vein injection. Kidneys were harvested at day 8 postinfection for determination of the number of CFU per gram of tissue. (B) BALB/c mice were shaved and challenged subcutaneously with a bolus of 1 × 107 CFU of wild-type or Δplc mutant S. aureus. A 10-mm punch biopsy specimen was excised from lesions at day 4 postinfection for determination of the number of CFU per gram of tissue. (C) The viability of Δplc strains exposed to mouse whole blood was determined by dividing the number of bacteria recovered after 3 h of exposure to the initial bacterial challenge. The numbers of CFU were determined by serial dilution and plating on TSA plates. The results in panel C represent the means ± SEMs for three independent experiments.
Tables
- TABLE 1
Strains and plasmids used in this study
Strain or plasmid Description or function Source or reference Strains E. coli strains DH5α Plasmid maintenance Protein Express TOP10 Cloning strain Invitrogen BL21 Star(DE3) Recombinant protein production Invitrogen S. aureus strains AH1263 USA300 CA-MRSA Erms (LAC*) 55 RN4220 Restriction deficient 56 MRSA252 USA200 MRSA 57 UAMS-1 Osteomyelitis isolate 58 Newman MSSA 59 COL HA-MRSA 60 MW2 USA400 CA-MRSA 61 NRS22 USA600 MRSA 62 TCH1516 USA300 CA-MRSA 63 JMB1145 LAC* SAUSA300_2337-38Δ This work JMB1148 LAC* SAUSA300_0217-18Δ This work JMB1219 LAC* SAUSA300_1219-20Δ This work JMB1223 LAC* SAUSA300_2035-36Δ This work JMB1232 LAC* SAUSA300_2558-59Δ This work JMB1241 LAC* SAUSA300_1798-99Δ This work JMB1357 LAC* SAUSA300_0254-55Δ This work JMB1358 LAC* SAUSA300_1638-39Δ This work JMB1359 LAC* SAUSA300_2308-09Δ This work JMB1377 LAC* SAUSA300_1865-66Δ This work JMB1383 LAC* SAUSA300_1307-08Δ This work JMB1293 LAC* SAUSA300_1441-42Δ This work AH1292 LAC* Δagr::tetM 64 AH1558 LAC* sae::spec 65 JMB1515 LAC* SAUSA300_0645-46Δ This work MJW44 LAC* SAUSA300_0099Δ(plc) This work MJW120 RN4220 Δplc This work MJW131 Newman Δplc This work Plasmids pJB38 Mutation generation vector 66 pCM28 Complementation vector 30 pET28a Protein expression vector Invitrogen pSKerm-MCS Complementation vector 67 pSK236 Shuttle vector 68 pET16b Protein expression vector Invitrogen pMW1 pJB38 SAUSA300_0099Δ This work pMW4 pSKerm-MCS SAUSA300_0099 This work pMW12 pET28a containing srrA This work pMW14 pSK236 containing scn promoter and plc This work pMW17 pET16b containing srrB kinase domain This work pMW20 pCM28 containing plc This work pCM29 pCM28 containing sGFP 30 pCR2.1 Cloning vector Invitrogen pJMB150 pJB38 SAUSA300_0217-18Δ This work pJMB155 pJB38 SAUSA300_2337-38Δ This work pJMB168 pJB38 SAUSA300_2035-36Δ This work pJMB170 pJB38 SAUSA300_1219-20Δ This work pJMB181 pJB38 SAUSA300_2558-59Δ This work pJMB183 pJB38 SAUSA300_1798-99Δ This work pJMB202 pJB38 SAUSA300_1307-08Δ This work pJMB204 pJB38 SAUSA300_1865-66Δ This work pJMB205 pJB38 SAUSA300_2308-09Δ This work pJMB210 pJB38 SAUSA300_0254-55Δ This work pJMB211 pJB38 SAUSA300_1638-39Δ This work pJMB229 pJB38 SAUSA300_1441-42Δ This work pJMB230 pJB38 SAUSA300_0645-46Δ This work
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Table S1. Oligonucleotides used in this study.
DOCX, 32K
- Supplemental file 2
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Fig. S1. Purification of recombinant PI-PLC.
EPS, 3.3M
- Supplemental file 3
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Fig. S2. Growth phase regulation of plc and srrA.
EPS, 3.5M
- Supplemental file 4
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Fig. S3. Initial characterization of S. aureus Δplc mutant.
EPS, 3.2M
- Supplemental file 5
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Fig. S4. Construction of an S. aureus ΔsrrAB strain that overexpresses PI-PLC.
EPS, 2.2M
- Supplemental file 6
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Legends to Fig. S1 to S4.
DOCX, 14K
- Supplemental file 1
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