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

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.

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.

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).

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).

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).

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).

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).