肺炎鏈球菌 (Streptococcus pneumoniae) 是造成兒童感染的重要致病菌，它不僅會引起中耳炎、鼻竇炎以及肺炎，更會引發致命性的腦膜炎以及敗血症。在對抗肺炎鏈球菌入侵及感染的免疫防禦中，補體活化作用扮演相當重要的角色。然而肺炎鏈球菌卻會藉由本身的毒性因子干擾補體活化，以達到致病的目的。目前已知在傳統補體活化途徑中，C4b結合蛋白質 (C4b-binding protein, C4BP) 扮演著調控補體活化的重要角色。有些細菌會利用結合C4b結合蛋白質，進而達到干擾補體活化以逃避免疫系統對細菌的攻擊，例如同屬於鏈球菌的化膿性鏈球菌 (Streptococcus pyogenes) 就會結合C4b結合蛋白質，藉此減少化膿性鏈菌受補體活化的攻擊。本研究中，我們藉由正常人血清與肺炎鏈球菌作用後，利用C4b結合蛋白質的抗體偵測及流式細胞儀的方法證實了C4b結合蛋白質與血清蛋白質S會以複合體的形式與肺炎鏈球菌結合。此外，結合上肺炎鏈球菌的C4b結合蛋白質仍具有抑制補體活化作用並減少補體C3蛋白質在細菌表面沈積。然而，結果亦顯示C4b結合蛋白質-蛋白質S複合體卻讓肺炎鏈球菌更易受到巨噬細胞的吞噬作用。臨床菌株的測試分析結果發現，單純鼻咽腔帶菌者的肺炎鏈球菌不會與C4b結合蛋白質結合，而部份造成侵襲性感染如腦膜炎及肺炎的細菌則與C4b結合蛋白質有不同程度的結合。吞噬作用的實驗亦顯示與C4b結合蛋白質有較高結合性的肺炎鏈球菌較易受到巨噬細胞的吞噬作用。由本研究的結果發現，C4b結合蛋白質在肺炎鏈球菌感染所造的先天性免疫活化反應中扮演重要的角色，尤其是在補體活化及吞噬作用。

Streptococcus pneumoniae is the most common etiological agent of pneumonia in children and is responsible for a broad spectrum of diseases, such as sinusitis, otitis media, meningitis, peritonitis, and bacteremia. Complement activation plays a pivotal role in immune defense. However, complement evasion has been used as a defense mechanism for S. pneumoniae to against innate immunity. Complement regulator C4b-binding protein (C4BP), a classical pathway complement inhibitor, is essential for regulating complement activation. Pathogens such as Streptococcus pyogenes could bind to C4BP and then cause interference on complement activation to escape from immune attack. In this study, we first demonstrated that serum proteins C4BP and protein S bound to S. pneumoniae using immunostaining followed by flow cytometry. Depleting protein S decreased the C4BP binding upon bacterial surface, indicating that C4BP and protein S bound to S. pneumoniae in a complex form. Notably, C4BP/protein S-binding S. pneumoniae showed a decrease in the activation of complement as demonstrated by the detection of C3 deposition. However, these bacteria were more susceptible to macrophage-mediated phagocytosis while depletion of protein S from serum decreased these effects. Studies further showed that clinical isolates obtained from the different infectious sites displayed variable binding affinity to C4BP, while none of colonization isolates bound to C4BP. Those clinical isolates, which have higher C4BP binding ability, were more easily engulfed by macrophages than those without C4BP binding. In summary, C4BP is involved in the innate immune activation of pneumococcal infection, especially the complement activation and phagocytosis.

Center for Disease Control and Prevention. (1997). Prevention of pneumococcal disease: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 46,1-24.
Center for Disease Control and Prevention. (2008). Invasive pneumococcal disease in children 5 years after conjugate vaccine introduction--eight states, 1998-2005. MMWR 57, 144-148.
AlonsoDeVelasco, E., Verheul, A.F., Verhoef, J., et al. (1995). Streptococcus pneumoniae: virulence factors, pathogenesis, and vaccines. Microbiological reviews 59, 591-603.
Anderson, H.A., Maylock, C.A., Williams, J.A., et al. (2003). Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nature immunology 4, 87-91.
Amano, A., Nakagawa, I., and Yoshimori, T. (2006). Autophagy in innate immunity against intracellular bacteria. Journal of biochemistry 140, 161-166.
Berggard, K., Johnsson, E., Mooi, F.R.,et al. (1997). Bordetella pertussis binds the human complement regulator C4BP: role of filamentous hemagglutinin. Infection and immunity 65, 3638-3643.
Bishof, N.A., Welch, T.R., and Beischel, L.S. (1990). C4B deficiency: a risk factor for bacteremia with encapsulated organisms. The Journal of infectious diseases 162, 248-250.
Blom, A.M., Villoutreix, B.O., and Dahlback, B. (2004). Complement inhibitor C4b-binding protein-friend or foe in the innate immune system? Molecular immunology 40, 1333-1346.
Brodeur, S.R., Angelini, F., Bacharier, L.B., et al. (2003). C4b-binding protein (C4BP) activates B cells through the CD40 receptor. Immunity 18, 837-848.
Brown, J.S., Hussell, T., Gilliland, S.M., et al. (2002). The classical pathway is the dominant complement pathway required for innate immunity to Streptococcus pneumoniae infection in mice. Proceedings of the National Academy of Sciences of the United States of America 99, 16969-16974.
Brueggemann, A.B., Griffiths, D.T., Meats, E., et al. (2003). Clonal relationships between invasive and carriage Streptococcus pneumoniae and serotype- and clone-specific differences in invasive disease potential. The Journal of infectious diseases 187, 1424-1432.
Coates, H., Thornton, R., Langlands, J., et al. (2008). The role of chronic infection in children with otitis media with effusion: evidence for intracellular persistence of bacteria. Otolaryngol Head Neck Surg 138, 778-781.
Dahlback, B. (1991). Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. Thrombosis and haemostasis 66, 49-61.
Dahlback, B., and Stenflo, J. (1981). High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein. Proceedings of the National Academy of Sciences of the United States of America 78, 2512-2516.
Dave, S., Brooks-Walter, A., Pangburn, M.K., et al. (2001). PspC, a pneumococcal surface protein, binds human factor H. Infection and immunity 69, 3435-3437.
Forsgren, A., Brant, M., Mollenkvist, A., et al. (2001). Isolation and characterization of a novel IgD-binding protein from Moraxella catarrhalis. J Immunol 167, 2112-2120.
Garcia de Frutos, P., Alim, R.I., Hardig, et al. (1994). Differential regulation of alpha and beta chains of C4b-binding protein during acute-phase response resulting in stable plasma levels of free anticoagulant protein S. Blood 84, 815-822.
Ghaffar, F., Barton, T., Lozano, et al. (2004). Effect of the 7-valent pneumococcal conjugate vaccine on nasopharyngeal colonization by Streptococcus pneumoniae in the first 2 years of life. Clin Infect Dis 39, 930-938.
Greiff, L., Erjefalt, I., Svensson, C., et al. (1993). Plasma exudation and solute absorption across the airway mucosa. Clinical physiology (Oxford, England) 13, 219-233.
Griffin, J.H., Gruber, A., and Fernandez, J.A. (1992). Reevaluation of total, free, and bound protein S and C4b-binding protein levels in plasma anticoagulated with citrate or hirudin. Blood 79, 3203-3211.
Hammitt, L.L., Bruden, D.L., Butler, J.C.,et al. (2006). Indirect effect of conjugate vaccine on adult carriage of Streptococcus pneumoniae: an explanation of trends in invasive pneumococcal disease. The Journal of infectious diseases 193, 1487-1494.
Hazelzet, J.A., de Groot, R., van Mierlo, et al. (1998). Complement activation in relation to capillary leakage in children with septic shock and purpura. Infection and immunity 66, 5350-5356.
Hummell, D.S., Swift, A.J., Tomasz, A., et al. (1985). Activation of the alternative complement pathway by pneumococcal lipoteichoic acid. Infection and immunity 47, 384-387.
Janoff, E.N., Fasching, C., Orenstein, J.M., et al. (1999). Killing of Streptococcus pneumoniae by capsular polysaccharide-specific polymeric IgA, complement, and phagocytes. The Journal of clinical investigation 104, 1139-1147.
Janulczyk, R., Iannelli, F., Sjoholm, A.G., et al. (2000). Hic, a novel surface protein of Streptococcus pneumoniae that interferes with complement function. The Journal of biological chemistry 275, 37257-37263.
Jarva, H., Janulczyk, R., Hellwage, J., et al. (2002). Streptococcus pneumoniae evades complement attack and opsonophagocytosis by expressing the pspC locus-encoded Hic protein that binds to short consensus repeats 8-11 of factor H. J Immunol 168, 1886-1894.
Jarva, H., Jokiranta, T.S., Wurzner, R., et al. (2003). Complement resistance mechanisms of streptococci. Molecular immunology 40, 95-107.
Jarva, H., Ram, S., Vogel, U., et al. (2005). Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis. J Immunol 174, 6299-6307.
Kadioglu, A., Weiser, J.N., Paton, J.C., et al. (2008). The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nature reviews 6, 288-301.
Kask, L., Trouw, L.A., Dahlback, B., et al. (2004). The C4b-binding protein-protein S complex inhibits the phagocytosis of apoptotic cells. The Journal of biological chemistry 279, 23869-23873.
Laval, C.B., de Andrade, A.L., Pimenta, F.C., et al. (2006). Serotypes of carriage and invasive isolates of Streptococcus pneumoniae in Brazilian children in the era of pneumococcal vaccines. Clin Microbiol Infect 12, 50-55.
Lin, W.J., Lo, W.T., Chou, C.Y., et al. (2006). Antimicrobial resistance patterns and serotype distribution of invasive Streptococcus pneumoniae isolates from children in Taiwan from 1999 to 2004. Diagnostic microbiology and infectious disease 56, 189-196.
Lo, W.T., Wang, C.C., Yu, C.M., et al. (2003). Rate of nasopharyngeal carriage, antimicrobial resistance and serotype of Streptococcus pneumoniae among children in northern Taiwan. Journal of microbiology, immunology, and infection 36, 175-181.
Munoz-Almagro, C., Jordan, I., Gene, A., et al. (2008). Emergence of invasive pneumococcal disease caused by nonvaccine serotypes in the era of 7-valent conjugate vaccine. Clin Infect Dis 46, 174-182.
Neeleman, C., Geelen, S.P., Aerts, P.C., et. al. (1999). Resistance to both complement activation and phagocytosis in type 3 pneumococci is mediated by the binding of complement regulatory protein factor H. Infection and immunity 67, 4517-4524.
Nordstrom, T., Blom, A.M., Forsgren, A., et al. (2004). The emerging pathogen Moraxella catarrhalis interacts with complement inhibitor C4b binding protein through ubiquitous surface proteins A1 and A2. J Immunol 173, 4598-4606.
O'Brien, K.L., Millar, E.V., Zell, E.R., et al. (2007). Effect of pneumococcal conjugate vaccine on nasopharyngeal colonization among immunized and unimmunized children in a community-randomized trial. The Journal of infectious diseases 196, 1211-1220.
Pangburn, M.K. (2000). Host recognition and target differentiation by factor H, a regulator of the alternative pathway of complement. Immunopharmacology 49, 149-157.
Persson, C.G., Erjefalt, I., Alkner, U., et al. (1991). Plasma exudation as a first line respiratory mucosal defence. Clin Exp Allergy 21, 17-24.
Poehling, K.A., Talbot, T.R., Griffin, M.R., et al. (2006). Invasive pneumococcal disease among infants before and after introduction of pneumococcal conjugate vaccine. JAMA 295, 1668-1674.
Radtke, A.L., and O'Riordan, M.X. (2006). Intracellular innate resistance to bacterial pathogens. Cellular Microbiology 8, 1720-1729.
Rezende, S.M., Simmonds, R.E., and Lane, D.A. (2004). Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S-C4b binding protein complex. Blood 103, 1192-1201.
Roy, S., Knox, K., Segal, S., et al. (2002). MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet 359, 1569-1573.
Singleton, R.J., Hennessy, T.W., Bulkow, L.R., et al. (2007). Invasive pneumococcal disease caused by nonvaccine serotypes among alaska native children with high levels of 7-valent pneumococcal conjugate vaccine coverage. JAMA 297, 1784-1792.
Siu, L.K., Chu, M.L., Ho, M., et al. (2002). Epidemiology of invasive pneumococcal infection in Taiwan: antibiotic resistance, serogroup distribution, and ribotypes analyses. Microbial drug resistance (Larchmont, NY 8, 201-208.
Thern, A., Stenberg, L., Dahlback, B., et al. (1995). Ig-binding surface proteins of Streptococcus pyogenes also bind human C4b-binding protein (C4BP), a regulatory component of the complement system. J Immunol 154, 375-386.
Tu, A.H., Fulgham, R.L., McCrory, M.A., et al. (1999). Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae. Infection and immunity 67, 4720-4724.
Watson, D.A., and Musher, D.M. (1990). Interruption of capsule production in Streptococcus pneumonia serotype 3 by insertion of transposon Tn916. Infection and immunity 58, 3135-3138.
Wooster, D.G., Maruvada, R., Blom, A.M., et al. (2006). Logarithmic phase Escherichia coli K1 efficiently avoids serum killing by promoting C4bp-mediated C3b and C4b degradation. Immunology 117, 482-493.
Yuste, J., Sen, A., Truedsson, L., et al. (2008). Impaired opsonization with C3b and phagocytosis of Streptococcus pneumoniae in sera from subjects with defects in the classical complement pathway. Infection and immunity 76, 3761-3770.