C-Reactive Protein Protects Mice against Pneumococcal Infection via Both Phosphocholine-Dependent and Phosphocholine-Independent Mechanisms

Abstract

The mechanism of action of C-reactive protein (CRP) in protecting mice against lethal Streptococcus pneumoniae infection is unknown. The involvement of the phosphocholine (PCh)-binding property of CRP in its antipneumococcal function previously has been explored twice, with conflicting results. In this study, using three different intravenous sepsis mouse models, we investigated the role of the PCh-binding property of CRP by employing a CRP mutant incapable of binding to PCh. The ability of wild-type CRP to protect mice against infection was found to differ in the three models; the protective ability of wild-type CRP decreased when the severity of infection was increased, as determined by measuring mortality and bacteremia. In the first animal model, in which we used 25 g of CRP and 10 7 CFU of pneumococci, both wild-type and mutant CRP protected mice against infection, suggesting that the protection was independent of the PCh-binding activity of CRP. In the second model, in which we used 25 g of CRP and 5 ؋ 10 7 CFU of pneumococci, mutant CRP was not protective while wild-type CRP was, suggesting that the protection was dependent on the PCh-binding activity of CRP. In the third model, in which we used 150 g of CRP and 10 7 CFU of pneumococci, mutant CRP was as protective as wild-type CRP, again indicating that the protection was independent of the PCh-binding activity of CRP. We conclude that both PCh-dependent and PCh-independent mechanisms are involved in the CRP-mediated decrease in bacteremia and the resulting protection of mice against pneumococcal infection. Infection with Streptococcus pneumoniae is one of the most common causes of community-acquired pneumonia and septicemia worldwide (reviewed in references 1-3). C-reactive protein (CRP) is a plasma protein whose level in the blood is dramatically increased in patients with S. pneumoniae infection (reviewed in references 4-8). In experiments using animal models, passively administered human CRP, transgenic human CRP, and murine CRP all have been shown to protect mice against lethal infection with S. pneumoniae, as determined by decreased bacteremia and increased survival (9-12). In murine models of infection involving passively administered human CRP, CRP exerted its protective effect only when injected 6 h before to 2 h after the infection but not when mice received CRP 24 h or 36 h postinfection (13,14). Thus, the CRP-mediated protection of mice requires the presence of CRP in the early stages of infection. However, the mechanism of the protective action of CRP in mice during the early stages of infection is not known.CRP binds to pneumococci in both serum and Ca 2ϩ -containing buffers (14). The binding of CRP to pneumococci is mediated via a Ca 2ϩ -dependent interaction between CRP and phosphocholine (PCh) residues present on C-polysaccharide (PnC) of the cell wall of pneumococci (15). Because PnC-complexed CRP activates the complement system in both human and mouse sera (4, 16; reviewed in reference 17), it has been proposed that the mechanism of th...

Functional Transformation of C-reactive Protein by Hydrogen Peroxide

“…CRP has been shown to protect mice against pneumococcal infection via both PCh-dependent and PCh-independent mechanisms (74,75). CRP slows atherosclerosis development in a mouse model with humanlike hypercholesterolemia (76).…”

Purification of recombinant C-reactive protein mutants

“…The mutations may even result in increased binding of CRP to PEt compared to that of WT CRP. For example, CRP mutant F66A/T76Y/E81A has been shown to possess PEt-binding activity several fold higher than that of WT CRP (Gang et al, 2015). These CRP mutants can be purified using PEt-conjugated Sepharose beads.…”

“…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.…”

“…Recombinant CRP mutants can be expressed in mammalian cells and, if expressed, can be purified from the cell culture media. However, while the methods to purify wild-type (WT) CRP are well established (Agrawal et al, 2001(Agrawal et al, , 2002Gang et al, 2015;Macintyre, 1988;Nunomura et al, 1990;Pepys et al, 1977Pepys et al, , 2012Riley and Coleman, 1970;Volanakis et al, 1978), different purification strategies are needed to purify various mutant forms of CRP if the mutant does not bind to either Ca 2+ or PCh. Although several expression systems have been employed to express recombinant CRP, including Leishmania tarentolae, Escherichia coli, Pichia pastoris and baculovirus-infected cell lines and Trichoplusia ni larvae (Dortay et al, 2011;Kilpatrick et al, 2012;Marnell et al, 1995;Potempa et al, 2015;Tanaka et al, 2002), in this article, we report the methods used to purify pentameric recombinant WT and mutant CRP expressed in and secreted by mammalian cells.…”