PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans

Abstract

Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in colonization and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was constructed in a pneumococcal TIGR4 background. When the mutant and wild type strains were compared for their adherence to D562 and A549 cell lines, a reduction in adherence by the mutant was observed (p = 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host Escherichia coli resulting in augmented adherence to D562 (p = 0.002) and A549 (p = 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p = 0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p = 0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.

Fig 1. Surface expression analysis of PcpA and pneumococcal adherence

A: Equal bacterial counts (2×10⁷) were taken, washed twice and incubated with PcpA specific monoclonal antibodies in the dilution of 1:1000 for 1 h at 4⁰C. Cultures were washed twice and incubated with anti mouse goat secondary antibodies in the dilution of 1:500 and incubated for 30 mins at room temperature. The surface expression was studied by flow cytometry by taking 20000 events. Histogram shows the surface expression of PcpA wild type TIGR4 and lack of expression on TIGR4 PcpA mutant. B: PcpA expression analysis by western blotting. After bacteria were cultured until mid-log phase, total cellular protein samples were prepared and separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-PcpA monoclonal antibody. Lane 1: marker, lane 2: pcpA mutant and lane 3: Wild type TIGR4. C: D562 cell line was infected with 200 MOI of wild type TIGR4 pneumococci and PcpA isogenic mutant. *** represents the difference in adherence of TIGR4 pneumococci with PcpA isogenic mutant as highly significant (p=0.002). The adherence of wild type pneumococci on D562 cells was normalized to 100% and reduction in the adherence of mutant is the representation of % reduction in adherence as compared to wild type adherence. The data represents the mean with Standard error (SEM) of three experiments in triplicates. (D): Adherence of TIGR4 wild type and PcpA isogenic mutants on A549 cell line. *** represents the difference in adherence of TIGR4 pneumococci with PcpA isogenic mutant as highly significant (p=0.002). The adherence of wild type pneumococci on A549 cells was normalized to 100% and reduction in the adherence of mutant is the representation of % reduction in adherence as compared to wild type adherence. The data represents the mean with Standard error (SEM) of three experiments in triplicates.

Fig 2. Surface expression analysis of PcpA on E coli and adherence

A: Equal bacterial counts (PcpA expressed and non expressed E coli) (2×10⁷) were taken, washed twice and incubated with PcpA specific monoclonal antibodies in the dilution of 1:1000 for 1 h at 4⁰C. Cultures were washed twice and incubated with anti mouse goat secondary antibodies in the dilution of 1:500 and incubated for 30 mins at room temperature. The surface expression was studied by flow cytometry by taking 20000 events. Histogram shows the surface expression of PcpA on IPTG induced E coli and lack of expression on uninduced E coli harboring recombinant plasmid. B: D562 cell line was infected with 200 MOI of PcpA expressed and non expressed E coli represents the difference in adherence of adherence between the PcpA expressed and non expressed E coli strains (p=0.002). *** represents the difference in adherence of PcpA surface expressed E coli and PcpA unexpressed E coli. The adherence of surface PcpA expressed E coli on D562 cells was normalized to 100% and reduction in the adherence of PcpA unexpressed E coli is the representation of % reduction in adherence as compared to PcpA expressing E coli. The data represents the mean with Standard error (SEM) of three experiments in triplicates. C: A549 cell line was infected with 200 MOI of PcpA expressed and nonexpressed E coli * represents the difference in adherence of adherence between the PcpA expressed and non expressed E coli strains (p=0.015).* represents the difference in adherence of PcpA surface expressed E coli and PcpA unexpressed E coli. The adherence of surface PcpA expressed E coli on A549 cells was normalized to 100% and reduction in the adherence of PcpA unexpressed E coli is the representation of % reduction in adherence as compared to PcpA expressing E coli. The data represents the mean with Standard error (SEM) of three experiments in triplicates.

Figure 3. Aggregation of labeled pneumococci in the presence of purified human IgG and Fab fragments

Pneumococci were incubated with varying concentrations (0.5-10 μg) of IgG and Fab fragments for 30 minutes at 37°C. 10,000 events were run for each sample, yielding three main populations of pneumococcal bacterial cells. Forward scatter of histograms: IgG treatment (upper panel, open histograms) caused bacterial aggregation at different concentrations whereas Fab treatment (lower panel, open histograms) did not. Solid (filled) histograms show negative control where pneumococci were treated with PBS only. Mean fluorescence index (MFI) of IgG and Fab treated pneumococci was compared using Non parametric Mann Whitney test (p<0.05). The mean fluorescence index (MFI) of IgG treated (1 μg IgG) pneumococcal aggregates (MFI: 19,257) was significantly higher than Fab treated (1 μg Fab) pneumococci (MFI: 6827) (p<0.05).

Figure 4. Adherence inhibition with Fab anti-PcpA depleted fragments

A: PcpA and CbpA specific Fab fragments were depleted from the total IgG Fab samples by incubating with 100ng of recombinant PcpA or CbpA. The data was first analysed by one way ANNOVA due to multiple comparisons and was found to be significant (p=0.024). Individual comparisons were made by non parametric Mann Whitney test. The depletion of PcpA and CbpA specific IgG Fab resulted in a significant increase in the pneumococcal adherence (p = 0.026, 0.015) on D562 cells. The adherence of wild type pneumococci on D562 cells was normalized to 100% and the decrease or increase in the adherence as a result of Fab treatment or the depletion of antigen specific Fab was normalized accordingly. The data represents the mean with Standard error (SEM) of three experiments in triplicates. B: The PcpA isogenic mutant was treated with total and PcpA, CbpA depleted Fab fragments. The data were analysed as explained above. The depletion of PcpA specific Fab did not affect adherence (p=0.40); however, CbpA depleted Fabs resulted in an increase in adherence of PcpA mutant pneumococci to D562 Cells. C: E coli expressing PcpA or control E coli (PcpA unexpressed) were treated with Fab fragments as described above and subsequently incubated with D562 cells in the MOI of 1:200. The adherence of E coli expressing PcpA decreased marginally (p=0.1) while the adherence E coli that did not express PcpA remained unchanged after Fab treatment (p=0.97).

Figure 5

Effect of recombinant PcpA on pneumococcal adherence blocking: Pneumococcal adherence was observed in the presence of pneumococcal proteins (PcpA, CbpA). At the concentration used in the adherence assays (100 ng), proteins alone did not inhibit adherence. Incubation of the D562 cells with a higher concentration (500 ng) of PcpA or CbpA did inhibit pneumococcal adherence significantly (p<0.05).