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Cellular Microbiology: Pathogen-Host Cell Molecular Interactions

Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils

Mark J. White, Jeffrey M. Boyd, Alexander R. Horswill, William M. Nauseef
B. A. McCormick, Editor
Mark J. White
aInflammation Program, Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
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Jeffrey M. Boyd
cDepartment of Biochemistry and Microbiology, Rutgers University, New Brunswick, New Jersey, USA
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Alexander R. Horswill
aInflammation Program, Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
bDepartment of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, Iowa, USA
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William M. Nauseef
aInflammation Program, Department of Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA
bDepartment of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, and Veterans Administration Medical Center, Iowa City, Iowa, USA
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B. A. McCormick
Roles: Editor
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DOI: 10.1128/IAI.01168-13
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  • FIG 1
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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
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    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

  • Figures
  • Additional Files
  • TABLE 1

    Strains and plasmids used in this study

    Strain or plasmidDescription or functionSource or reference
    Strains
        E. coli strains
            DH5αPlasmid maintenanceProtein Express
            TOP10Cloning strainInvitrogen
            BL21 Star(DE3)Recombinant protein productionInvitrogen
        S. aureus strains
            AH1263USA300 CA-MRSA Erms (LAC*)55
            RN4220Restriction deficient56
            MRSA252USA200 MRSA57
            UAMS-1Osteomyelitis isolate58
            NewmanMSSA59
            COLHA-MRSA60
            MW2USA400 CA-MRSA61
            NRS22USA600 MRSA62
            TCH1516USA300 CA-MRSA63
            JMB1145LAC* SAUSA300_2337-38ΔThis work
            JMB1148LAC* SAUSA300_0217-18ΔThis work
            JMB1219LAC* SAUSA300_1219-20ΔThis work
            JMB1223LAC* SAUSA300_2035-36ΔThis work
            JMB1232LAC* SAUSA300_2558-59ΔThis work
            JMB1241LAC* SAUSA300_1798-99ΔThis work
            JMB1357LAC* SAUSA300_0254-55ΔThis work
            JMB1358LAC* SAUSA300_1638-39ΔThis work
            JMB1359LAC* SAUSA300_2308-09ΔThis work
            JMB1377LAC* SAUSA300_1865-66ΔThis work
            JMB1383LAC* SAUSA300_1307-08ΔThis work
            JMB1293LAC* SAUSA300_1441-42ΔThis work
            AH1292LAC* Δagr::tetM64
            AH1558LAC* sae::spec65
            JMB1515LAC* SAUSA300_0645-46ΔThis work
            MJW44LAC* SAUSA300_0099Δ(plc)This work
            MJW120RN4220 ΔplcThis work
            MJW131Newman ΔplcThis work
    Plasmids
        pJB38Mutation generation vector66
        pCM28Complementation vector30
        pET28aProtein expression vectorInvitrogen
        pSKerm-MCSComplementation vector67
        pSK236Shuttle vector68
        pET16bProtein expression vectorInvitrogen
        pMW1pJB38 SAUSA300_0099ΔThis work
        pMW4pSKerm-MCS SAUSA300_0099This work
        pMW12pET28a containing srrAThis work
        pMW14pSK236 containing scn promoter and plcThis work
        pMW17pET16b containing srrB kinase domainThis work
        pMW20pCM28 containing plcThis work
        pCM29pCM28 containing sGFP30
        pCR2.1Cloning vectorInvitrogen
        pJMB150pJB38 SAUSA300_0217-18ΔThis work
        pJMB155pJB38 SAUSA300_2337-38ΔThis work
        pJMB168pJB38 SAUSA300_2035-36ΔThis work
        pJMB170pJB38 SAUSA300_1219-20ΔThis work
        pJMB181pJB38 SAUSA300_2558-59ΔThis work
        pJMB183pJB38 SAUSA300_1798-99ΔThis work
        pJMB202pJB38 SAUSA300_1307-08ΔThis work
        pJMB204pJB38 SAUSA300_1865-66ΔThis work
        pJMB205pJB38 SAUSA300_2308-09ΔThis work
        pJMB210pJB38 SAUSA300_0254-55ΔThis work
        pJMB211pJB38 SAUSA300_1638-39ΔThis work
        pJMB229pJB38 SAUSA300_1441-42ΔThis work
        pJMB230pJB38 SAUSA300_0645-46ΔThis work

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Table S1. Oligonucleotides used in this study.

      DOCX, 32K

    • Supplemental file 2 -

      Fig. S1. Purification of recombinant PI-PLC.

      EPS, 3.3M

    • Supplemental file 3 -

      Fig. S2. Growth phase regulation of plc and srrA.

      EPS, 3.5M

    • Supplemental file 4 -

      Fig. S3. Initial characterization of S. aureus Δplc mutant.

      EPS, 3.2M

    • Supplemental file 5 -

      Fig. S4. Construction of an S. aureus ΔsrrAB strain that overexpresses PI-PLC.

      EPS, 2.2M

    • Supplemental file 6 -

      Legends to Fig. S1 to S4.

      DOCX, 14K

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Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
Mark J. White, Jeffrey M. Boyd, Alexander R. Horswill, William M. Nauseef
Infection and Immunity Mar 2014, 82 (4) 1559-1571; DOI: 10.1128/IAI.01168-13

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Phosphatidylinositol-Specific Phospholipase C Contributes to Survival of Staphylococcus aureus USA300 in Human Blood and Neutrophils
Mark J. White, Jeffrey M. Boyd, Alexander R. Horswill, William M. Nauseef
Infection and Immunity Mar 2014, 82 (4) 1559-1571; DOI: 10.1128/IAI.01168-13
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