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Host Response and Inflammation

Differential Role of CbpA and PspA in Modulation of In Vitro CXC Chemokine Responses of Respiratory Epithelial Cells to Infection with Streptococcus pneumoniae

Rikki M. A. Graham, James C. Paton
Rikki M. A. Graham
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia
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James C. Paton
School of Molecular and Biomedical Science, University of Adelaide, Adelaide, South Australia 5005, Australia
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  • For correspondence: james.paton@adelaide.edu.au
DOI: 10.1128/IAI.00954-06
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  • FIG. 1.
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    FIG. 1.

    Western blot analysis of CbpA and PspA mutants. Lysates of the indicated strains were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto nitrocellulose, and reacted with mouse polyclonal antiserum specific for CbpA (A and C) or PspA (B and D). A band of the appropriate size for CbpA (∼95 kDa) was seen in the lysate of WT D39, while the sizes of bands seen in the lysates of the domain mutants were consistent with deletion of the specific domains (∼85 kDa in each case). Lysates of WT D39 and pspA domain mutants were separated by SDS-PAGE, electroblotted onto nitrocellulose, and reacted with mouse polyclonal antiserum specific for PspA (as described in Materials and Methods). A band of the appropriate size for PspA (∼80 kDa) was seen in the lysate of WT D39, while the sizes of bands seen in the lysates of the domain mutants were consistent with deletion of the specific domains (∼65 kDa for the PspAΔh1, PspAΔh2, and PspAΔpro mutants and ∼50 kDa for the PspAΔhelix mutant).

  • FIG. 2.
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    FIG. 2.

    Schematic representation of domain deletion mutants. (A) CbpA mutants. The leader sequence at the N terminus is hatched; other domains are designated as follows: Hyp, hypervariable region; SR1, small repeat region 1; SR2, small repeat region 2; Pro, proline-rich region; CBD, choline-binding domain. The numbers below the WT D39 CbpA map denote amino acid residues. (B) PspA mutants. The leader sequence at the N terminus is hatched; other domains are designated as follows: regions 1 and 2, respective portions of the α-helical domain; Pro, proline-rich region; CBD, choline-binding domain. The numbers denote amino acid residues in WT D39 PspA.

  • FIG. 3.
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    FIG. 3.

    CXC chemokine mRNA response of respiratory epithelial cells after infection with S. pneumoniae D39. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were infected with 5 × 107 CFU S. pneumoniae D39 for 2 or 4 h, at which time cellular RNA was extracted and analyzed by real-time RT-PCR, using oligonucleotides specific for IL-8, MIP-2α, ENA-78, MGSA, MIP-2β, and GCP-2. GAPDH mRNA was used as an internal control, and results are expressed as increases (n-fold) in mRNA at 2 or 4 h relative to an uninfected 0-h control.

  • FIG. 4.
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    FIG. 4.

    CXC chemokine mRNA response of respiratory epithelial cells to S. pneumoniae D39, ΔCbpA mutant, or ΔPspA mutant. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were incubated with approximately 5 × 107 CFU S. pneumoniae D39, ΔCbpA mutant, or ΔPspA mutant for 2 or 4 h before extraction of cellular RNA and analysis of chemokine-specific mRNA by real-time RT-PCR. Results are expressed as increases (n-fold) of chemokine mRNA relative to a 0-h control. Experiments were performed in quadruplicate and analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (relative to D39 at the respective time point).

  • FIG. 5.
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    FIG. 5.

    IL-8 secretion from respiratory epithelial cells infected with WT D39, the ΔCbpA mutant, or the ΔPspA mutant. Cell culture supernatants from A549 (A) and Detroit-562 (B) cells infected with 5 × 107 CFU D39, ΔCbpA mutant, or ΔPspA mutant were assayed for IL-8 by ELISA. The data shown are the means ± SEs from three independent experiments. Results were analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. **, P < 0.01; *, P < 0.05 (relative to D39).

  • FIG. 6.
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    FIG. 6.

    CXC chemokine mRNA response of respiratory epithelial cells to CbpA domain mutants. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were incubated with 5 × 107 CFU S. pneumoniae D39 or otherwise isogenic mutants, with in-frame deletions of regions encoding specific domains of CbpA, for 4 h before extraction of total cellular RNA and analysis of chemokine mRNA by real-time RT-PCR. Data are means ± SEs from three independent experiments. Data were analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. ***, P < 0.001; **, P < 0.01; *, P < 0.05 (relative to D39).

  • FIG. 7.
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    FIG. 7.

    IL-8 secretion from respiratory epithelial cells in response to CbpA domain mutants. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were incubated with S. pneumoniae D39 or CbpA domain mutants for 4 h before collection of the cell culture supernatant and analysis of IL-8 by ELISA. Data are means ± SEs from three independent experiments. Data were analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. ***, P < 0.001; **, P < 0.01 (relative to D39).

  • FIG. 8.
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    FIG. 8.

    CXC chemokine mRNA response of respiratory epithelial cells to PspA domain mutants. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were incubated with 5 × 107 CFU S. pneumoniae D39 or otherwise isogenic mutants, with in-frame deletions of specific domains of PspA, for 4 h before extraction of cellular RNA and analysis of chemokine mRNA by real-time RT-PCR with specific oligonucleotides. Data are means ± SEs from three independent experiments. Results were analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. *, P < 0.05 (relative to D39).

  • FIG. 9.
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    FIG. 9.

    IL-8 secretion from respiratory epithelial cells infected with PspA domain mutants. Confluent monolayers of A549 (A) or Detroit-562 (B) cells were incubated with 5 × 107 CFU WT S. pneumoniae D39 or PspA domain mutants for 4 h before collection of the cell culture supernatant and analysis of IL-8 by ELISA. Data are means ± SEs from three independent experiments. Data were analyzed for statistical significance by one-way ANOVA with a post hoc Bonferroni test. **, P < 0.01; *, P < 0.05 (relative to D39).

Tables

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  • TABLE 1.

    S. pneumoniae strains used in this study

    StrainDescriptionSource or reference
    D39Serotype 2 (NCTC 7466) 1
    CbpA−D39 CbpA insertion-duplication mutant, Eryr 4
    ΔCbpA cbpA deletion mutant of D39This study
    CbpAΔHypHypervariable region of cbpA (nt 424-720) deletion mutant of D39This study
    CbpAΔSR1Small repeat region 1 of cbpA (nt 721-1206) deletion mutant of D39This study
    CbpAΔSR2Small repeat region 2 of cbpA (nt 1207-1548) deletion mutant of D39This study
    CbpAΔProProline-rich region of cbpA (nt 1549-1761) deletion mutant of D39This study
    PspA−D39 PspA insertion-duplication mutant 27
    pspA::erm mutant pspA erm insertion mutant of D39This study
    ΔPspA pspA deletion mutant of D39This study
    PspAΔh1α-Helical region 1 of pspA (nt 210-657) deletion mutant of D39This study
    PspAΔh2α-Helical region 2 of pspA (nt 658-1083) deletion mutant of D39This study
    PspAΔhelixα-Helical region of pspA (nt 210-1083) deletion mutant of D39This study
    PspAΔproProline-rich region of pspA (nt 1084-1329) deletion mutant of D39This study
  • TABLE 2.

    Oligonucleotides used in this study

    OligonucleotideSequence (5′→3′)aLocation (accession no.)
    IDPspAaACAAGTCTAGCCAGCGTCGCTD39 pspA nt 151-171 (M74122)
    IDPspAbTATCTGATACTTTGAACCATTGGCD39 pspA complementary nt 1874-1897
    RMAG1aatttaatgggcTTACTTATTCATCTAAATTTACCTCTTTTR6 genome complementary nt 128339-128367 (NC_003098)
    RMAG2tagatgaataagTAAGCCGATTAAATTAAAGCATGTTAAR6 genome nt 130213-130239
    RMAG3TTAGAACGGCTTAAAATCAGATATGAR6 genome nt 126448-126473
    RMAG4CGCCACCTAGAACACTCTTCGR6 genome complementary nt 131668-131687
    RMAG5ataaacatgtttGTAAACTAAACCTAATATAACTAGTTAR6 genome complementary nt 1987525-1987552
    RMAG6ttaggtttagtttaCAAACATGTTTATTTCCTTCTATATR6 genome nt 1989641-1989667
    RMAG7GCTGCACCGATAGACAGACGCR6 genome nt 1985081-1985101
    RMAG8TCCTTGACCATATCTGCTCACCR6 genome complementary nt 1991426-1991447
    RMAG12tgaagaagtcgctGAAGGAGTGATTACATGAACAApVA891 ery nt 5103-5125
    RMAG13tggctcttcagcCTCATAGAATTATTTCCTCCCGpVA891 ery nt 4363-4384
    RMAG14attcactccttcAGCGACTTCTTCAGCATCCACD39 pspA complementary nt 718-738
    RMAG15taattctatgagGCTGAAGAGCCATCGCAACCAD39 pspA nt 1075-1095
    PspA Helix1 1tttagcttcttcTGCTCTTACAACAGTAGGCTGD39 pspA complementary nt 199-209
    PspA Helix1 2gttgtaagagcaGAAGAAGCTAAAGCAAAATTAGAAD39 pspA nt 658-678
    PspA Helix2 1tggtttttctggTAGTTTTTTAGTAAGTTCTGGTGCD39 pspA complementary nt 634-657
    PspA Helix2 2actaaaaaactaCCAGAAAAACCAGCTCCAGCTD39 pspA nt 1084-1104
    PspA Pro 1tttccagcctgtCTCATTAACTGCTTTCTTAAGGTCD39 pspA complementary nt 1063-1083
    PspA Pro 2gcagttaatgagACAGGCTGGAAACAAGAAAACGD39 pspA nt 1330-1350
    PspA helix 1tggtttttctggTGCTCTTACAACAGTAGGCTGD39 pspA complementary nt 199-209
    PspA helix 2gttgtaagagcaCCAGAAAAACCAGCTCCAGCTD39 pspA nt 1084-1104
    CbpA hyp1cttttctcctggCGCATGAACCACACTTCCCATD39 cbpA complementary nt 403-423 (AF068646)
    CbpA hyp2gtggttcatgcgCCAGGAGAAAAGGTAGCAGAAD39 cbpA nt 721-741
    CbpA sm rep1 1cttttttcctgaTTTCAATGTATCTTTTTTAAACTTCTCD39 cbpA complementary nt 694-720
    CbpA sm rep1 2gatacattgaaaTCAGGAAAAAAGGTAGCAGAAGCTD39 cbpA nt 1207-1230
    CbpA sm rep2 1ttgttcagctggTTTCAGGGATGAGCTTGGAAGD39 cbpA complementary nt 1186-1206
    CbpA sm rep2 2tcatccctgaaaCCAGCTGAACAACCACAACCAD39 cbpA nt 1549-1569
    CbpA Pro 1ttgtttccagccTTTTTCTTTAACTTTATCTTCTTCTGD39 cbpA complementary nt 1523-1548
    CbpA Pro 2gttaaagaaaaaGGCTGGAAACAAGAAAACGGTD39 cbpA nt 1762-1782
    IL-8 FwdGAAGGAACCATTCTCACTGTGTGTAIL-8 mRNA nt 75-99 (M28130)
    IL-8 RevTTATGAATTCTCAGCCCTCTTCAAAAACIL-8 mRNA complementary nt 402-375
    ENA-78 FwdGAACCCGCGACCGCTCGCENA-78 mRNA nt 62-79 (XM_003507)
    ENA-78 RevAGAAAAGGGGCTTCTGGATCAAENA-78 mRNA complementary nt 393-372
    GCP-2 FwdCTCCACCCAGCTCAGGAACCGCP-2 mRNA nt 14-33 (XM_003502)
    GCP-2 RevGAAAAGGGGCTTCCGGGTCCAGCP-2 mRNA complementary nt 351-331
    MGSA FwdAGCCACACTCAAGAATGGGCGMSGA mRNA nt 304-324 (XM_003504)
    MGSA RevTGGCATGTTGCAGGCTCCTCMSGA mRNA complementary nt 758-777
    MIP-2α FwdATTTGTTAATATTTCTTCGTGATGACATATCAMIP-2α mRNA nt 709-740 (X53799)
    MIP-2α RevTCGAAACCTCTCTGCTCTAACACMIP-2α mRNA complementary nt 1010-1032
    MIP-2β FwdAGAACATCCAAAGTGTGAATGTAAGGMIP-2β mRNA nt 198-223 (X53800)
    MIP-2β RevTCCTTTCCAGCTGTCCCTAGAAMIP-2β mRNA complementary nt 458-479
    GAPDH FwdTCCTTGGAGGCCATGTGGGCCATGAPDH mRNA nt 206-228 (XM_033258)
    GAPDH RevTGATGACATCAAGAAGGTGGTGAAGGAPDH mRNA complementary nt 445-421
    • ↵ a Nucleotides in lowercase are 5′ extensions enabling annealing of PCR products.

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Differential Role of CbpA and PspA in Modulation of In Vitro CXC Chemokine Responses of Respiratory Epithelial Cells to Infection with Streptococcus pneumoniae
Rikki M. A. Graham, James C. Paton
Infection and Immunity Nov 2006, 74 (12) 6739-6749; DOI: 10.1128/IAI.00954-06

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Differential Role of CbpA and PspA in Modulation of In Vitro CXC Chemokine Responses of Respiratory Epithelial Cells to Infection with Streptococcus pneumoniae
Rikki M. A. Graham, James C. Paton
Infection and Immunity Nov 2006, 74 (12) 6739-6749; DOI: 10.1128/IAI.00954-06
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KEYWORDS

Bacterial Proteins
Chemokines, CXC
Respiratory Mucosa
Streptococcus pneumoniae

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