Effects of PspA and antibodies to PspA on activation and deposition of complement on the pneumococcal surface

Abstract

Streptococcus pneumoniae infection is a frequent cause of pneumonia, otitis media, meningitis, and septicemia. Pneumococcal surface protein A (PspA) is an important virulence factor on the pathogen surface, and it is known to interfere with complement activation. In this study, flow cytometry was used to study the effects of PspA and antibodies to PspA on the deposition of complement C3 on the surface of a capsular type 3 strain, WU2, and its PspA- mutant, JY1119. Using naive mouse serum as a complement source, measurable deposition of C3 was observed within 4 min on PspA- pneumococci, and the amount of surface-bound C3 accumulated rapidly as the amount of serum was increased. In contrast, very little C3 was deposited on the PspA+ strain. In nonimmune mouse serum, the classical pathway was the dominant activation pathway triggered by PspA- pneumococci. Accordingly, EGTA blocked almost all of the complement activation. Moreover, a significant amount of C3 was still deposited on the PspA- strain when serum from factor B-deficient mice was used. This deposition was not observed on the PspA+ pneumococci, indicating that PspA may inhibit complement deposition via the classical pathway. Furthermore, under the conditions we tested, PspA also inhibited C3 deposition when the classical pathway was initiated by antibodies to capsular polysaccharide. Antibodies to PspA could overcome the anticomplementary effect of PspA, allowing for increased complement activation and C3 deposition onto PspA+ bacteria.

FIG. 1.

C3 deposition on the surfaces of PspA⁺ and PspA⁻ strains. WU2 (PspA⁺) and JY1119 (PspA⁻) were incubated at 37°C for 30 min with 10% naive CBA/N mouse serum (NMS) diluted with GVB²⁺, 10% NMS with 10 mM EDTA, or 10% heat-inactivated (HI) serum diluted with GVB²⁺. The buffer control samples were incubated with GVB²⁺ instead of 10% serum. The percentage of C3-positive bacteria (intensity greater than 10 on axis of FL1) is indicated for each sample. The data shown are representative of at least three experiments.

FIG. 2.

Influence of serum concentrations on C3 deposition. Different concentrations of NMS (from naive CBA/N mice) diluted with GVB²⁺ were incubated with PspA⁺ or PspA⁻ pneumococci. In each case, the bacteria were incubated with serum for 30 min at 37°C. Error bars indicate the standard errors of three experiments.

FIG. 3.

Time course of C3 deposition. PpsA⁺ and PspA⁻ pneumococci were incubated with 10% NMS from CBA/N mice, and the reactions were stopped at various time points using ice-cold PBS buffer containing 10 mM EDTA. The mean fluorescence intensity for a representative experiment (of three) is shown.

FIG. 4.

Complement activation and C3 deposition onto pneumococci via the alternative pathway. PspA⁺ and PspA⁻ strains were incubated with 30% NMS from CBA/N mice in the presence of Ca²⁺ and Mg²⁺ or in the presence of EGTA (blocking the activation of the classical/MBL pathway) and Mg²⁺. The result of a representative experiment of three is shown.

FIG. 5.

Complement activation and C3 deposition in the absence of the alternative pathway. Pneumococci were incubated with 10% serum from C57BL/6J WT or FB^−/− mice. The result of a representative experiment of three is shown.

FIG. 6.

Effect of PspA on C3 deposition via the classical pathway triggered by anticapsule MAb. WU2 (PspA⁺) and JY1119 (PspA⁻) were pretreated with different amounts of anticapsule MAb, washed, and then incubated with 10% FB^−/− (A) or WT (FB^+/+) (B) serum diluted with GVB²⁺. The data are representative of three experiments. (C) The amount of anticapsule MAb bound on PspA⁺ and PspA⁻ strains. WU2 (PspA⁺) and JY1119 (PspA⁻) were incubated with different dilutions of anticapsule MAb, and the surface binding of MAb was detected with Fluorescein isothiocyanate-conjugated goat anti-mouse IgG. Error bars indicate the standard errors of duplicate samples.

FIG. 7.

Comparison of C3 deposition on PspA⁺ and PspA⁻ pneumococci incubated with pre- or postimmune serum against recombinant PspA protein. (A) Pneumococci were incubated with 10% pre- or postimmune serum from CBA/N mice. (B) Pneumococci were pretreated with 10% pre- or postimmune serum from CBA/N mice in the presence of EDTA, washed, and then incubated with 10% pooled naive NMS.

FIG. 8.

Effect of antibodies to PspA on C3 deposition on PspA⁺ pneumococci. Strain WU2 was pretreated with different concentrations of pre- (open symbols) or postimmune (filled symbols) serum against recombinant PspA from CBA/N mice (in the presence of EDTA), washed, and then incubated with 10% serum from C57BL/6J FB^−/− (circle) or WT (square) mice. Data are presented as mean intensities ± standard errors of the means of duplicate samples in one of three different experiments.