研究生: |
曾渝婷 Tseng, Yu-Ting |
---|---|
論文名稱: |
K1大腸桿菌的NlpI蛋白幫助細菌結合C4bp使其逃脫血清中典型補體路徑所調控的擊殺 NlpI of Escherichia coli K1 contributes to the recruitment of C4b binding protein to evade serum killing mediated by the classical complement pathway |
指導教授: |
鄧景浩
Teng, Ching-Hao |
學位類別: |
碩士 Master |
系所名稱: |
醫學院 - 分子醫學研究所 Institute of Molecular Medicine |
論文出版年: | 2010 |
畢業學年度: | 98 |
語文別: | 英文 |
論文頁數: | 57 |
中文關鍵詞: | K1大腸桿菌 、NlpI蛋白 、C4b結合蛋白 、補體活化 |
外文關鍵詞: | Escherichia coli K1, NlpI, C4b-binding protein (C4bp), the complement activation |
相關次數: | 點閱:80 下載:6 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
K1大腸桿菌(Escherichia coli K1)是造成革蘭氏陰性菌新生兒腦膜炎的主要致病菌。K1大腸桿菌所引發的新生兒腦膜之發展通常需要透過血液的散佈與轉移。因此,細菌在血流中存活且增殖進而引起菌血症是新生兒腦膜炎的致病機制中很重要的步驟。然而現今對於K1大腸桿菌造成菌血症相關的致病因子尚未被了解透徹。在對抗外來微生物入侵與感染的宿主免疫之中,補體系統扮演著很重要的角色。然而,為了要成功感染人體,微生物必須發展出一些策略來躲避補體的攻擊。文獻指出細菌可黏附血清中的C4b結合蛋白(C4b-binding protein, C4bp)以干擾補體的活化,進而逃離其攻擊。根據先前的動物實驗數據顯示,在老鼠血液中,臨床分離出的K1大腸桿菌RS218之nlpI突變株與野生株比較之後,其生存能力呈現顯著的下降。因此,本研究主要探討NlpI如何貢獻於K1大腸桿菌生存在血流中的機制。目前我們發現,當nlpI 基因剔除之後會降低K1大腸桿菌在血清中的生存能力,然而,在補體蛋白被高溫去活性的血清中,nlpI 基因剔除則不影響K1大腸桿菌在其中的生存。在與血清作用後,補體蛋白之活化而沉積在nlpI突變株表面的程度顯著的高於野生株。這些結果顯示NlpI蛋白之功能與K1大腸桿菌抵抗補體系統之攻擊有關。此外,nlpI突變株在補體之傳統路徑去活化的血清當中,其生存能力恢復似野生株,顯示NlpI的表達可幫助K1大腸桿菌抵抗補體系統中傳統路徑所活化的攻擊。另外,當K1大腸桿菌之NlpI蛋白被刪除之後會降低K1大腸桿菌黏附C4b 結合蛋白到自身表面的能力。藉由比較野生株與nlpI突變株在正常人類血清與C4b結合蛋白被去除之血清的生長程度,我們也確認了nlpI突變株與C4b結合蛋白結合能力之下降,與其在血清中無法抵抗補體擊殺而生存能力下降的現象相關。
由本研究的結果發現一個新穎的細菌因子「NlpI蛋白」,其可幫助K1大腸桿菌逃離血清的擊殺,主要的機制在於此細菌因子的表達可促使C4b結合蛋白被K1大腸桿菌黏附進而抑制了補體系統中的傳統路徑之活化。
E. coli K1 is the most common organism associated with neonatal Gram-negative meningitis. Neonatal E. coli K1 meningitis usually develops as a result of hematogenous spread. Therefore, survival and multiplication in the blood is a prerequisite for E. coli K1 to further infect the central nervous system. Complement system plays a pivotal role in host defense against infection. To survive in the blood, bacteria need to develop a strategy of complement evasion. C4b binding protein (C4bp), an inhibitor of the classical and the lectin complement pathways, has been reported to be recruited to the surface of pathogenic bacteria to interfere activation of the complement system. Our preliminary data have shown that deletion of the nlpI gene of E. coli K1 decreased the bacterium’s ability to cause high-degree bacteremia in 5-day-old rats, suggesting that NlpI is involved in E. coli K1 survival in the blood stream. This study aims to investigate how NlpI contributes to E. coli K1 survival in the blood stream.
We found that deletion of nlpI decreased the survival of E. coli K1 in the normal human serum but did not affect the bacterial survival in the heat-inactivated serum in which the complement system was inactivated. In addition, after incubation with serum, the nlpI mutant exhibited significantly higher levels of C3 and MAC (membrane attack complex) deposition than did the wild-type strain. These results demonstrated that NlpI is involved in E. coli K1 evasion of complement attack. The nlpI mutant showed similar survival with the wild-type strain in the modified human serum in which the classical complement pathway was blocked, suggesting that NlpI is involved in E. coli K1 evasion of attack by the classical complement pathway.
In addition, deletion of nlpI decreased the ability of E. coli K1 to recruit C4bp to the bacterial surface. By comparing the survival of the wild-type and the nlpI mutant strains in the normal and C4bp-depleted sera, we demonstrated that the decreased C4bp binding on the nlpI mutant is responsible for its decreased resistance to serum-mediated killing.
In conclusion, we found that NlpI, a novel bacterial factor, is involved in E. coli K1 evasion of serum-mediated attack. This bacterial factor contributes to E. coli K1 serum survival through facilitating C4bp deposition on bacterial surface to block the attack by the classical complement pathway.
Accardo, P., P. Sanchez-Corral, et al. (1996). "Binding of human complement component C4b-binding protein (C4BP) to Streptococcus pyogenes involves the C4b-binding site." J Immunol 157(11): 4935-9.
Achtman, M., A. Mercer, et al. (1983). "Six widespread bacterial clones among Escherichia coli K1 isolates." Infect Immun 39(1): 315-35.
Barbosa, A. S., P. A. Abreu, et al. (2009). "Immune evasion of leptospira species by acquisition of human complement regulator C4BP." Infect Immun 77(3): 1137-43.
Barnich, N., M. A. Bringer, et al. (2004). "Involvement of lipoprotein NlpI in the virulence of adherent invasive Escherichia coli strain LF82 isolated from a patient with Crohn's disease." Infect Immun 72(5): 2484-93.
Berggard, K., E. Johnsson, et al. (1997). "Bordetella pertussis binds the human complement regulator C4BP: role of filamentous hemagglutinin." Infect Immun 65(9): 3638-43.
Berggard, K., E. Johnsson, et al. (2001). "Binding of human C4BP to the hypervariable region of M protein: a molecular mechanism of phagocytosis resistance in Streptococcus pyogenes." Mol Microbiol 42(2): 539-51.
Blom, A. M. (2002). "Structural and functional studies of complement inhibitor C4b-binding protein." Biochem Soc Trans 30(Pt 6): 978-82.
Blom, A. M., K. Berggard, et al. (2000). "Human C4b-binding protein has overlapping, but not identical, binding sites for C4b and streptococcal M proteins." J Immunol 164(10): 5328-36.
Blom, A. M., L. Kask, et al. (2001). "Structural requirements for the complement regulatory activities of C4BP." J Biol Chem 276(29): 27136-44.
Dahlback, B. and J. Stenflo (1981). "High molecular weight complex in human plasma between vitamin K-dependent protein S and complement component C4b-binding protein." Proc Natl Acad Sci U S A 78(4): 2512-6.
Das, A. K., P. W. Cohen, et al. (1998). "The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein-protein interactions." Embo J 17(5): 1192-9.
Datsenko, K. A. and B. L. Wanner (2000). "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products." Proc Natl Acad Sci U S A 97(12): 6640-5.
Dietzman, D. E., G. W. Fischer, et al. (1974). "Neonatal Escherichia coli septicemia-- bacterial counts in blood." J Pediatr 85(1): 128-30.
Dieudonne-Vatran, A., S. Krentz, et al. (2009). "Clinical isolates of Streptococcus pneumoniae bind the complement inhibitor C4b-binding protein in a PspC allele-dependent fashion." J Immunol 182(12): 7865-77.
Grimwood, K., P. Anderson, et al. (2000). "Twelve year outcomes following bacterial meningitis: further evidence for persisting effects." Arch Dis Child 83(2): 111-6.
Guo, R. F. and P. A. Ward (2005). "Role of C5a in inflammatory responses." Annu Rev Immunol 23: 821-52.
Haines, J. L., M. A. Hauser, et al. (2005). "Complement factor H variant increases the risk of age-related macular degeneration." Science 308(5720): 419-21.
Hallstrom, T., A. M. Blom, et al. (2009). "Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance." J Immunol 183(4): 2593-601.
Hallstrom, T., H. Jarva, et al. (2007). "Interaction with C4b-binding protein contributes to nontypeable Haemophilus influenzae serum resistance." J Immunol 178(10): 6359-66.
Harvey, D., D. E. Holt, et al. (1999). "Bacterial meningitis in the newborn: a prospective study of mortality and morbidity." Semin Perinatol 23(3): 218-25.
Haupt, K., M. Reuter, et al. (2008). "The Staphylococcus aureus protein Sbi acts as a complement inhibitor and forms a tripartite complex with host complement Factor H and C3b." PLoS Pathog 4(12): e1000250.
Hessing, M., R. A. Vlooswijk, et al. (1990). "The localization of heparin-binding fragments on human C4b-binding protein." J Immunol 144(1): 204-8.
Howard, C. J. and A. A. Glynn (1971). "The virulence for mice of strains of Escherichia coli related to the effects of K antigens on their resistance to phagocytosis and killing by complement." Immunology 20(5): 767-77.
Jann, K. and B. Jann (1992). "Capsules of Escherichia coli, expression and biological significance." Can J Microbiol 38(7): 705-10.
Jarva, H., S. Ram, et al. (2005). "Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis." J Immunol 174(10): 6299-307.
Kaper, J. B., J. P. Nataro, et al. (2004). "Pathogenic Escherichia coli." Nat Rev Microbiol 2(2): 123-40.
Kim, B. Y., J. Kang, et al. (2005). "Invasion processes of pathogenic Escherichia coli." Int J Med Microbiol 295(6-7): 463-70.
Kim, K. S. (2008). "Mechanisms of microbial traversal of the blood-brain barrier." Nat Rev Microbiol 6(8): 625-34.
Kim, K. S., J. H. Kang, et al. (1988). "Functional activities of monoclonal antibodies to the O side chain of Escherichia coli lipopolysaccharides in vitro and in vivo." J Infect Dis 157(1): 47-53.
Kirjavainen, V., H. Jarva, et al. (2008). "Yersinia enterocolitica serum resistance proteins YadA and ail bind the complement regulator C4b-binding protein." PLoS Pathog 4(8): e1000140.
Kohl, J. (2006). "Self, non-self, and danger: a complementary view." Adv Exp Med Biol 586: 71-94.
Lamb, J. R., S. Tugendreich, et al. (1995). "Tetratrico peptide repeat interactions: to TPR or not to TPR?" Trends Biochem Sci 20(7): 257-9.
Lambris, J. D., D. Ricklin, et al. (2008). "Complement evasion by human pathogens." Nat Rev Microbiol 6(2): 132-42.
Oglesby, T. J., C. J. Allen, et al. (1992). "Membrane cofactor protein (CD46) protects cells from complement-mediated attack by an intrinsic mechanism." J Exp Med 175(6): 1547-51.
Ohara, M., H. C. Wu, et al. (1999). "Identification and characterization of a new lipoprotein, NlpI, in Escherichia coli K-12." J Bacteriol 181(14): 4318-25.
Okroj, M., D. Heinegard, et al. (2007). "Rheumatoid arthritis and the complement system." Ann Med 39(7): 517-30.
Parker, C. J. (2001). "Isolation and functional assay of the membrane complement inhibitors CD55 (DAF) and CD59 (MIRL)." Curr Protoc Immunol Chapter 13: Unit 13 5.
Pietikainen, J., T. Meri, et al. "Binding of the complement inhibitor C4b-binding protein to Lyme disease Borreliae." Mol Immunol 47(6): 1299-305.
Pluschke, G. and M. Achtman (1984). "Degree of antibody-independent activation of the classical complement pathway by K1 Escherichia coli differs with O antigen type and correlates with virulence of meningitis in newborns." Infect Immun 43(2): 684-92.
Pluschke, G., J. Mayden, et al. (1983). "Role of the capsule and the O antigen in resistance of O18:K1 Escherichia coli to complement-mediated killing." Infect Immun 42(3): 907-13.
Pluschke, G., A. Mercer, et al. (1983). "Induction of bacteremia in newborn rats by Escherichia coli K1 is correlated with only certain O (lipopolysaccharide) antigen types." Infect Immun 39(2): 599-608.
Prasadarao, N. V., A. M. Blom, et al. (2002). "A novel interaction of outer membrane protein A with C4b binding protein mediates serum resistance of Escherichia coli K1." J Immunol 169(11): 6352-60.
Preissner, K. T. (1991). "Structure and biological role of vitronectin." Annu Rev Cell Biol 7: 275-310.
Ram, S., M. Cullinane, et al. (2001). "Binding of C4b-binding protein to porin: a molecular mechanism of serum resistance of Neisseria gonorrhoeae." J Exp Med 193(3): 281-95.
Rautemaa, R. and S. Meri (1999). "Complement-resistance mechanisms of bacteria." Microbes Infect 1(10): 785-94.
Robbins, J. B., G. H. McCracken, Jr., et al. (1974). "Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis." N Engl J Med 290(22): 1216-20.
Scheufler, C., A. Brinker, et al. (2000). "Structure of TPR domain-peptide complexes: critical elements in the assembly of the Hsp70-Hsp90 multichaperone machine." Cell 101(2): 199-210.
Schnaitman, C. A. (1970). "Protein composition of the cell wall and cytoplasmic membrane of Escherichia coli." J Bacteriol 104(2): 890-901.
Schwalbe, R. A., B. Dahlback, et al. (1990). "Independent association of serum amyloid P component, protein S, and complement C4b with complement C4b-binding protein and subsequent association of the complex with membranes." J Biol Chem 265(35): 21749-57.
Sjoberg, A. P., L. A. Trouw, et al. (2009). "Complement activation and inhibition: a delicate balance." Trends Immunol 30(2): 83-90.
Stevens, J. P., M. Eames, et al. (2003). "Long term outcome of neonatal meningitis." Arch Dis Child Fetal Neonatal Ed 88(3): F179-84.
Tadokoro, A., H. Hayashi, et al. (2004). "Interaction of the Escherichia coli lipoprotein NlpI with periplasmic Prc (Tsp) protease." J Biochem (Tokyo) 135(2): 185-91.
Tadokoro, A., H. Hayashi, et al. (2004). "Interaction of the Escherichia coli lipoprotein NlpI with periplasmic Prc (Tsp) protease." J Biochem 135(2): 185-91.
Teng, C. H., Y. Xie, et al. (2006). "Effects of ompA deletion on expression of type 1 fimbriae in Escherichia coli K1 strain RS218 and on the association of E. coli with human brain microvascular endothelial cells." Infect Immun 74(10): 5609-16.
Teng, C. H., Y. T. Tseng, et al. (2010). "NlpI contributes to Escherichia coli K1 strain RS218 interaction with human brain microvascular endothelial cells." Infect Immun 78(7): 3090-6.
Terai, I., K. Kobayashi, et al. (1993). "Human serum mannose binding protein (MBP): development of an enzyme-linked immunosorbent assay (ELISA) and determination of levels in serum from 1085 normal Japanese and in some body fluids." Biochem Med Metab Biol 50(1): 111-9.
Trouw, L. A., H. M. Nielsen, et al. (2008). "C4b-binding protein in Alzheimer's disease: binding to Abeta1-42 and to dead cells." Mol Immunol 45(13): 3649-60.
Trouw, L. A., S. C. Nilsson, et al. (2005). "C4b-binding protein binds to necrotic cells and DNA, limiting DNA release and inhibiting complement activation." J Exp Med 201(12): 1937-48.
Vidarsson, G. and J. G. van de Winkel (1998). "Fc receptor and complement receptor-mediated phagocytosis in host defence." Curr Opin Infect Dis 11(3): 271-8.
Wei, Y., R. B. Ota, et al. (1996). "Determination of human plasma and leukocyte ascorbic acid by microtiter plate assay." J. Nutr. Biochem. 7(3): 179-186.
Wilson, C. G., T. Kajander, et al. (2005). "The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold." FEBS J 272(1): 166-79.
Wyle, F. A., M. S. Artenstein, et al. (1972). "Immunologic response of man to group B meningococcal polysaccharide vaccines." J Infect Dis 126(5): 514-21.
Yao, Y., Y. Xie, et al. (2006). "Genomic comparison of Escherichia coli K1 strains isolated from the cerebrospinal fluid of patients with meningitis." Infect Immun 74(4): 2196-206.
Zhou, J., M. I. Fonseca, et al. (2008). "Complement C3 and C4 expression in C1q sufficient and deficient mouse models of Alzheimer's disease." J Neurochem 106(5): 2080-92.
Zinkernagel, R. M. (2001). "Maternal antibodies, childhood infections, and autoimmune diseases." N Engl J Med 345(18): 1331-5.
Zipfel, P. F., R. Wurzner, et al. (2007). "Complement evasion of pathogens: common strategies are shared by diverse organisms." Mol Immunol 44(16): 3850-7.