Human C-reactive protein protects mice from Streptococcus pneumoniae infection without binding to pneumococcal C-polysaccharide

Abstract

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcgamma receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.

FIGURE 1

Survival curves of mice infected with S. pneumoniae. Mice were injected with 10⁸ CFU of bacteria, with or without 150 μg of either WT, or F66A, or F66A/E81A CRP. CRP was injected first; bacteria were injected 30 min later. Deaths were recorded twice per day for 10 days. The data are combined from three separate experiments with six mice in each group: Experiment 1 was performed with six male mice in each of group A, B, and C; Experiment 2 was performed with six male mice in each group A, B, and D; Experiment 3 was performed with six female mice in each group A, B, and D. The p values for the differences in the survival curves among groups A and B, A and C, and A and D are 0.0001, 0.001, and 0.0001, respectively. The p values for the differences in the survival curves among groups B and C, B and D, and C and D are 0.33, 0.42, and 0.73, respectively.

FIGURE 2

Bacteremia in mice treated with WT and mutant CRP. Blood samples were collected from each surviving mouse shown in Fig. 1 for the first 5 days postinfection. Bacteremia was determined by plating. Each dot represents one mouse. The horizontal line in each group of mice represents the median value of bacteremia in that group. A bacteremia value of >10⁷ indicates a dead mouse. The p values for the differences among groups A and B, A and C, and A and D, on days 2, 3, and 4 are <0.008. The p values for the differences among groups B and C, B and D, and C and D on days 2, 3, and 4 are >0.05.

FIGURE 3

Binding of WT and mutant CRP to PnC. A representative experiment is shown. A, Deposition of C3 reflecting the formation of CRP-PnC complexes as a function of CRP dose. Microtiter wells were coated with PnC. Increasing concentrations of purified CRP were then added to the wells. Normal human serum was then added as the source of complement. Deposited C3 was detected by using anti-human C3d mAb. Values on the y-axis represent binding of anti-C3d to C3 deposited on PnC-CRP complexes. B, Binding of CRP to killed strain WU2 bacteria. Microtiter wells were coated with killed bacteria. Increasing concentrations of purified CRP were then added to the wells. Bound CRP was detected using anti-CRP mAb HD2.4 as a reporter. C, Binding of CRP by live S. pneumoniae strain R36A. A fixed number of bacteria were grown in the presence of increasing amount of CRP, as shown on the x-axis. Then, CRP that bound to bacteria was eluted and measured by ELISA. The amount of bound CRP is shown on the y-axis.

FIGURE 4

Binding of CRP by live virulent S. pneumoniae strain WU2. A, Binding in Todd-Hewitt broth. A fixed number of live bacteria were grown in the presence of increasing amount of CRP, as shown on the x-axis. Then, CRP that bound to bacteria was eluted and measured by ELISA. Amount of bound CRP is shown on the y-axis. B, Binding in mouse serum. A fixed number of live bacteria were grown in the presence of 2000 ng of WT or mutant CRP. Then, CRP that bound to bacteria was eluted and measured by ELISA. The amount of bound CRP is shown on the y-axis.

FIGURE 5

Effect of endotoxin on the CRP-mediated protection of mice infected with S. pneumoniae. Mice were injected with 0.5 × 10⁸ CFU bacteria, with 150 μg of either WT or F66A/E81A CRP. Control groups A and B received 150 μl of TBS. The endotoxin levels (EU) are given in the parentheses. TBS or CRP was injected first; bacteria were injected 30 min later. Deaths were recorded twice per day for 10 days. Six male mice were used in all groups. The p values for the differences in the survival curves among groups A and B, A and C, A and D, and C and D are 0.30, 0.01, 0.004, and 0.87, respectively.

FIGURE 6

Survival curves of mice infected with S. pneumoniae. Mice were injected with 10⁸ CFU bacteria, either alone (●), or with 150 μg of WT CRP. CRP was administered either 30 min before infection (■) or 36 h after infection (□). Six male mice were used in all groups. Deaths were recorded twice per day for 10 days.