Human C-Reactive Protein Protects Mice fromStreptococcus pneumoniaeInfection 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 Fcγ 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.

Purification of recombinant C-reactive protein mutants

“…Polyclonal anti-CRP antibody is purified from rabbit anti-human CRP antiserum (Sigma-Aldrich) by a separate affinity chromatography using a CRP-conjugated Sepharose column. The conjugation of CRP or anti-CRP antibody to Sepharose beads can be performed using the AminoLink Immobilization kit (Pierce) according to manufacturer's instructions (Agrawal et al, 2002;Suresh et al, 2007). The capacity of the anti-CRP-Sepharose beads to bind CRP per ml of packed beads is determined by using a known amount of purified WT CRP.…”

“…The significance of the recognition function of CRP in its alternate structural pentameric conformation in host defense is unknown. However, ex vivo and in vivo experiments in animal models of inflammatory diseases indicate that CRP exerts, although limited, anti-pneumococcal, anti-atherosclerotic, anti-arthritic and antiamyloidogenic functions Agrawal et al, 2008Agrawal et al, , 2010Chang et al, 2012;Gang et al, 2012Gang et al, , 2015Jiang et al, 2006;Jones et al, 2011;Kovacs et al, 2007;Nayeri et al, 2010;Ngwa et al, 2016;Ozawa et al, 2016;Simons et al, 2014;Singh et al, 2008;Suresh et al, 2006Suresh et al, , 2007Szalai et al, 1995;Yother et al, 1982). CRP has also been shown to alleviate experimental autoimmune encephalomyelitis (Zhang et al, 2015).…”

“…For 2002; Gang et al, 2012Gang et al, , 2015Suresh et al, 2006Suresh et al, , 2007. Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media.…”

C-Reactive Protein-Bound Enzymatically Modified Low-Density Lipoprotein Does Not Transform Macrophages into Foam Cells

“…The method of construction, expression, and some biochemical characterization of the CRP mutants have been described earlier (14,33).…”

“…Native CRP was purified from pleural fluid as described earlier (33,34). Purified CRP was stored in TBS at Ϫ80°C.…”