Complement Activation by C-Reactive Protein Is Critical for Protection of Mice Against Pneumococcal Infection

Abstract

C-reactive protein (CRP), a component of the innate immune system, is an antipneumococcal plasma protein. Human CRP has been shown to protect mice against infection with lethal doses of Streptococcus pneumoniae by decreasing bacteremia. in vitro, CRP binds to phosphocholine-containing substances, such as pneumococcal C-polysaccharide, in a Ca²⁺-dependent manner. Phosphocholine-complexed human CRP activates the complement system in both human and murine sera. The mechanism of antipneumococcal action of CRP in vivo, however, has not been defined yet. In this study, we tested a decades-old hypothesis that the complement-activating property of phosphocholine-complexed CRP contributes to protection of mice against pneumococcal infection. Our approach was to investigate a CRP mutant, incapable of activating murine complement, in mouse protection experiments. We employed site-directed mutagenesis of CRP, guided by its three-dimensional structure, and identified a mutant H38R which, unlike wild-type CRP, did not activate complement in murine serum. Substitution of His³⁸ with Arg in CRP did not affect the pentameric structure of CRP, did not affect the binding of CRP to pneumococci, and did not decrease the stability of CRP in mouse circulation. Employing a murine model of pneumococcal infection, we found that passively administered H38R CRP failed to protect mice against infection. Infected mice injected with H38R CRP showed no reduction in bacteremia and did not survive longer, as opposed to infected mice treated with wild-type CRP. Thus, the hypothesis that complement activation by phosphocholine-complexed CRP is an antipneumococcal effector function was supported. We can conclude now that complement activation by phosphocholine-complexed CRP is indeed essential for CRP-mediated protection of mice against pneumococcal infection.

Keywords: C-reactive protein; acute phase response; complement; inflammation; pneumococcal infection.

Figure 1

Activation of murine complement by human CRP. A representative of three experiments is shown. (A) Binding of CRP to P-PnC. Microtiter wells were coated with P-PnC. CRP diluted in TBS-Ca was added to the wells. Bound CRP was detected by using an anti-CRP antibody. Color was developed and the OD was read at 405 nm. (B) Activation of murine C3 by CRP complexed with P-PnC. Microtiter wells were coated with P-PnC. CRP diluted in TBS-Ca was added to the wells. Normal mouse serum was then added to the wells. Deposited C3 was detected by using goat anti-mouse C3 antibody. Color was developed and the OD was read at 405 nm.

Figure 2

Overall pentameric structure of H38R CRP. A representative of three experiments is shown. (A) Elution profiles of CRP from the gel filtration column are shown. CRP in TBS containing 2 mM CaCl₂ was applied to the column and eluted with the same buffer. Sixty fractions (0.25 ml) were collected and protein measured (A₂₈₀) to determine the elution volume of CRP. (B) Denaturing SDS-PAGE of CRP. A Coomassie brilliant blue-stained gel (lane 2, WT CRP; lane 3, H38R CRP), is shown. (C) Binding of CRP to pneumococci. Microtiter wells were coated with pneumococci. CRP diluted in TBS-Ca was added to the wells. Bound CRP was detected by using an anti-CRP antibody. Color was developed and the OD was read at 405 nm.

Figure 3

Clearance of H38R CRP from mouse circulation. Mice were injected with 50 μg of CRP. Blood was collected at various time points, sera separated, and the concentration of CRP measured.

Figure 4

Survival curves of mice infected with pneumococci with and without CRP. CRP was injected first; pneumococci were injected 30 min later. The data are combined from two separate experiments with seven mice in each group in each experiment. The p-values for the differences in the survival curves between groups A B, A D, B C, and C D were <0.05. The p-values for the differences in the survival curves between groups A C and B D were >0.05.

Figure 5

Bacteremia in mice infected with pneumococci with and without CRP. (A) Blood was collected from each surviving mouse shown in Figure 4. Bacteremia was determined by plating. Each dot represents one mouse. The horizontal line in each group of mice represents median bacteremia. A bacteremia value of >10⁸ indicates a dead mouse. The p-values for the differences between groups A B and A D were <0.05. The p value for the difference between groups A C was >0.05. (B) The median bacteremia values for each group shown in (A) are plotted.