Pneumolysin-induced complement depletion during experimental pneumococcal bacteremia

Abstract

To quantify complement depletion by pneumolysin during Streptococcus pneumoniae bacteremia, cirrhotic and control rats were infected intravenously with one of three isogenic mutant strains of S. pneumoniae expressing different forms of pneumolysin. Outcome measures included clearance of the organisms from the bloodstream, alterations in 50% serum hemolytic complement (CH(50)) activity and complement C3 levels during infection, and serum opsonic capacity at 18 h postinfection. Cirrhotic rats had significantly lower CH(50) and C3 levels than control rats, both before and after infection. However, initial complement levels did not predict bacterial load after 18 h of infection. Changes in CH(50) and C3 levels over the 18-h period correlated with numbers of H+C+ but not H+C- or PLY- organisms in the bloodstream at 18 h postinfection. The sera of cirrhotic rats infected with the H+C+ strain had significantly decreased levels of C3 and showed significantly lower opsonizing activity for S. pneumoniae than sera from H+C+-infected control rats. These studies suggest that under limiting concentrations of complement, the expression of pneumolysin by pneumococci has a significant, negative effect on serum complement levels and reduces serum opsonic activity.

FIG. 1

Clearance of mutant bacterial strains from the bloodstreams of control (A) and cirrhotic (B) rats. Control and cirrhotic rats (n = 10 and 13, respectively, for rats infected with the H+C+ strain and 6 for all other groups) were infected intravenously (i.v.) with one of the isogenic mutant strains at 1 × 10⁷ to 5 × 10⁷ CFU/ml of peripheral blood, as verified within the first 5 min postinfection. Plate counts also were performed on blood collected at 2 and 18 h postinfection to determine clearance. Data presented are means ± standard deviations (SD [error bars]). Significant differences in clearance of the various strains by cirrhotic rats, as determined by the Kruskal-Wallace test, are indicated by brackets. ∗, value is significantly higher for cirrhotic rats than for control rats (P = 0.008); ∗∗, value is significantly lower for cirrhotic rats than for control rats (P = 0.02), as determined by the Mann-Whitney U test.

FIG. 2

Comparison of serum CH₅₀ activity levels (A) and serum C3 levels (B) in cirrhotic and control rats. All rats (n = 21 or 25/time point) were infected i.v. with one of the isogenic mutant strains at 1 × 10⁷ to 5 × 10⁷ CFU/ml of their peripheral blood. Data shown are medians, with the range from the 25th to the 75th percentile shown for each time point. ∗, value significantly lower in cirrhotic versus control rats (P = 0.0001, 0.001, and 0.04 before infection, 2 h postinfection, and 18 h postinfection, respectively). #, value significantly lower in cirrhotic versus control rats (P = 0.02 and 0.006 before infection and 18 h postinfection, respectively).

FIG. 3

Comparative alterations in CH₅₀ activity levels (A) and C3 levels (B) during infection with isogenic mutant strains. All rats were bled 2 days before infection (pre-infect) and again 18 h postinfection with 1 × 10⁷ to 5 × 10⁷ CFU of one of the isogenic mutant strains per ml of peripheral blood. Postinfection groups are labeled with the organism strain designation (n = 9 cirrhotic and 13 control animals for the H+C+ strain and 6 each for the H+C− and PLY− strains). Serum CH₅₀ activity levels and C3 levels shown are medians, with the range from the 25th to the 75th percentile indicated. Significant differences (indicated by bars) between pre- and postinfection values were calculated by the Mann-Whitney U test. Differences among 18-h values for rats in the same treatment group infected with different strains were calculated by the Kruskal-Wallis test.

FIG. 4

Correlation between bacteremia counts at 18 h postinfection and the percent change in serum CH₅₀ activity levels (A) and C3 levels (B) of rats infected with the H+C+ strain. Each graph includes values for 9 cirrhotic and 13 control rats infected with 1 × 10⁷ to 5 × 10⁷ CFU of the H+C+ strain per ml of blood. Correlations between variables were determined by calculation of Spearman's rank order coefficient (r_s).