Title

克雷白氏肺炎桿菌生物膜在致病機轉中所扮演的角色

Translated Titles

Role of Biofilm in The Pathogenic Mechanism of Klebsiella pneumoniae

Authors

莊恕函

Key Words

克雷白氏肺炎桿菌 ; Klebsiella pneumoniae

PublicationName

中山醫學大學生化微生物免疫研究所學位論文

Volume or Term/Year and Month of Publication

2013年

Academic Degree Category

碩士

Advisor

詹明修;賴怡琪

Content Language

繁體中文

Chinese Abstract

克雷白氏肺炎桿菌為具莢膜的革蘭氏陰性兼性厭氧桿菌,是人體口腔、皮膚及腸道中的常在菌叢,卻也是社區感染及院內感染重要的病原菌之一,主要會導致肺炎、急性膽管炎、尿道感染及肝膿瘍,主要的毒力因子包括有表面抗原 (莢膜與脂多醣)、黏附因子 (線毛) 及鐵螯合系統,近年來由克雷白氏肺炎桿菌造成的肝膿瘍病患身上發現到,以外科手術方式除去膿瘍後仍然會復發,推測是克雷白氏肺炎桿菌的其他毒力因子如生物膜所造成。生物膜是細菌附著於含水環境表面或組織上聚集後,所分泌之液態聚基質包裹菌體,形成一種鬆散且易漂動的狀態。由於在生物膜中的細菌呈現休眠的狀態,因此對抗生素敏感性降低,且在生物膜的保護下也不易受到宿主免疫系統的偵測及攻擊,故被視為是體內慢性感染的主因。在這篇論文中,我們假設生物膜能夠增加克雷白氏肺炎桿菌的致病能力,因此在比較分離自肝膿瘍病患及非肝膿瘍病患的克雷白氏肺炎桿菌生物膜形成能力後,發現生物膜形成能力與致病力並不成正比,且每株臨床菌株對於養分及氧氣需求皆不同,因此無法定義何種環境對於生物膜的形成是有幫助的。另一方面,我們也自這些臨床菌株中挑選出生物膜形成能力最強的菌株KLA1242,以跳躍子突變法篩選到幾個與生物膜形成相關的基因,也進一步證實第三型線毛對生物膜形成能力是重要的。後續實驗中我們使用了標準菌株CG43研究生物膜與免疫細胞的交互作用,將小鼠脾臟細胞分別與CG43的菌液和生物膜共培養,觀察細胞活化的情況及各種細胞激素的分泌量,為求更精確定量生物膜及其活性,我們使用懸浮態菌液及生物膜萃取出的多醣與脾臟細胞共培養。在本篇的研究中我們發現T細胞、B細胞及巨噬細胞皆會受到活化,尤其是B細胞和巨噬細胞活化程度約為30%~50%,另外活化的細胞分泌的細胞激素有IL-1β、IL-2、IL-6、IL-10、TNF-α及IFN-γ,特別是IL-6和IL-10表現穩定且有隨著多醣濃度增加而分泌量提高,我們推測多醣引起的反應一開始可能為發炎反應,分泌IL-6來活化其他免疫細胞,而後期轉而分泌IL-10活化較多的B細胞來製造抗體。另外我們以TLR2小鼠來分析多醣是否會透過TLR2來活化免疫細胞,我們發現TLR2剔除的細胞活化比例顯著較正常小鼠脾臟細胞來的低,TLR2剔除脾臟細胞活化後分泌的細胞激素量與正常小鼠脾臟細胞活化後比較有顯著的減少,大部分的細胞激素幾乎沒有分泌,故我們證明多醣的確是透過TLR2來活化免疫細胞。在台灣,由克雷白氏肺炎桿菌導致的肝膿瘍病患通常患有糖尿病,為了解糖尿病個體的免疫細胞對生物膜多醣的反應,我們進一步以STZ誘導的糖尿病小鼠來看在高糖環境下的免疫細胞對於多醣刺激的反應,發現糖尿病鼠脾臟細胞在IL-6、IL-10、TNF-α、TGF-β及IFN-γ表現量上確實較正常鼠來得低。雖然這些結果無法證明生物膜在感染上是否扮演關鍵的角色,但我們可以知道莢膜多醣與生物膜多醣主要透過TLR2以及其他受體使B細胞與巨噬細胞大量活化,並誘使IL-6與IL-10穩定且大量的表現來調控免疫反應。

English Abstract

Klebsiella pneumoniae (KP) is a Gram negative facultative anaerobic bacilli with capsule, and normally in oral cavity, skin and intestinal flora of human. It is one of the community-acquired and nosocomial infections, and mainly causes pneumonia, acute cholangitis, urinary tract infection and liver abscess. The major virulence factors of KP include surface antigen (capsular lipopolysaccharide), adhesion factor (fimbriae) and iron acquisition system etc. In recent years, it has been found that replapses of patients with Klebsiella pneumoniae-induced liver abscess often occurr after an operation for removing of abscess. It is proposed causing by other virulence factors such as biofilm. The biofilm will be built by bacteria those secrete the liquid polysaccharide matrix after bacteria attached on the surface of the aqueous environment or tissues, and then parcel bacteria form a loose and easy to wandering state. Because of rendering dormant state in biofilms, bacteria are more tolerant to antibiotics. Furthermore, biofilm protects bacteria from being detected and attecked by immune system, and passes for the main cause of chronic infection in vivo. In the thesis, we hypothesize that biofilm could increase the pathogenicity of KP too. We compared the biofilm-forming abilities of KP strains obtained from patients with liver abscess with strains from patients with abscess not in liver. We found that there was no correlation between bacterial virulence and biofilm-forming ability. We also found that requirements of nutrients and oxygen of different strains were very diverse. So we could not conclude what environmental factor(s) will be helpful for biofilm biognenesis. We selected the KLA1242 strain with the most ability of biofilm formation from those clinical isolates for screening what gene(s) may be involved in biofilm formation by transposome mutagenesis method, and our results demonstrated that the type 3 pili is important for biofilm formation. In further studies, the planktonic of and biofilm from KP standard strain CG43 were cocultured with murine splenocytes respectively for studying the interacting outcome of biofilm with immune cells by quantitating cell activation and cytokine-secreting levels. For more precisely quantifying the biofilm and its bioactivity, the biofilm polysaccharide extracted by KP was be used and the capsule polysaccharide from planktonic bacteria was as a control. Our results demonstraed that immune cells incoulding T cells, B cells and macrophages could be activated by polysaccharide and especially the percentages of activating stages of B cells and macrophages were high at 30%~50%. The polysaccharide treatment could also induce secretions of IL-1β, IL-2, IL-6, IL-10, TNF-α and IFN-γ and especially the levels of IL-6 and IL-10 were polysaccharide dose-dependent. We suggest that IL-6 may help inflammatory responses in the beginning of infection, and the following IL-10 secretion may sustain antibody production. For understanding whether TLR2 will be the receptor of biofilm polysaccharide, the splenocytes obtained from TLR2 mice were treated with biofilm polysaccharide, and our results indicated that the cell activations and levels of cytokines produced by splenocytes from TLR2 mice were significantly less than biofilm polysaccharide-treated splenocytes from wild-type mice. Our results proved that biofilm polysaccharide activate immune cells via TLR2 receptor signaling. In Taiwan, patients with liver abscess caused by KP usually also suffer with diabetes. For understanding the immune responses of individuals with diabetes to biofilm polysaccharide, splenocytes from STZ-induced diabetic mice were treated with polysaccharide under a high glucose condition. Our results demonstrated that levels of L-6, IL-10, TNF-α,TGF-β and IFN-γ secreated by polysaccharide-treated splenocytes from diabetic mice were significantly lower than from wild-type mice. Although our results could not demonstrate the role of biofilm in KP infection in vivo, but we prove the capsular polysaccharide and biofilm polysaccharide could activate immune cells, especially B cells and macrophages, mainly through TLR2 and partial through other receptor. Furthermore, the large-scale productions of IL-6 and IL-10 by biofilm polysaccharide-stimulated immune cells may contribute to regulation of immune responses.

Topic Category 醫藥衛生 > 基礎醫學
醫學院 > 生化微生物免疫研究所
Reference
  1. Akira, S., Takeda, K., and Kaisho, T. 2001. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2(8):675-680.
    連結:
  2. Angelini, A., Cendron, L., Goncalves, S., Zanotti, G., and Terradot, L. 2008. Structural and enzymatic characterization of HP0496, a YbgC thioesterase from Helicobacter pylori. Proteins 72(4):1212-1221.
    連結:
  3. Beutler, B., Jiang, Z., Georgel, P., Crozat, K., Croker, B., Rutschmann, S., Du, X., and Hoebe, K. 2006. Genetic Analysis of Host Resistance: Toll-Like Receptor Signaling and Immunity at Large. Annu Rev Immunol 24:353-89.
    連結:
  4. Chan, Y. R., Liu, J. S., Pociask, D. A., Zheng, M., Mietzner, T. A., Berger T., Mak, T. W., Clifton, M. C., Strong, R. K., and Ray, P., et al. 2009. Lipocalin 2 is required for pulmonary host defense against Klebsiella infection. J Immunol 182(8):4947-4956.
    連結:
  5. Chen, S. C., Yen, C. H., Lai, K. C., Tsao, S. M., Cheng, K. S., Chen, C. C., Lee, M. C., and Chou, M. C. 2005. Pyogenic liver abscesses with Escherichia coli: etiology, clinical course, outcome, and prognostic factors. Wien Klin Wochenschr 117(23-24):809-815.
    連結:
  6. Costerton, J. W., Stewart, P. S., and Greenberg, E. P. 1999. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 284(5418):1318-1322.
    連結:
  7. Devaraj S., Tobias P., Kasinath B. S., Ramsamooj R., Afify A., and Jialal I. 2011. Knockout of toll-like receptor-2 attenuates both the proinflammatory state of diabetes and incipient diabetic nephropathy. Arterioscler Thromb Vasc Biol 31(8):1796-804.
    連結:
  8. Ellis E.M. 2002. Microbial aldo-keto reductases. FEMS Immunol Med Microbiol 26(3-4):259-65.
    連結:
  9. Fang, F. C., Sandler, N., and Libby, and S.J. 2005. Liver abscess caused by magA+ Klebsiella pneumoniae in North America. J Clin Microbiol 43(2): 991-992.
    連結:
  10. Flo, T.H., Smith, K.D., Sato, S., Rodriguez, D.J., Holmes, M.A., Strong, R.K., Akira, S., and Aderem, A. 2004. Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432(7019):917-921.
    連結:
  11. Fung, C. P., Chang, F. Y., Lin, J. C., Ho, D.M. T., Chen, C. T., Chen, J. H., Yeh, K. M., Chen, T. L., Lin, Y. T., and Siu, L. K. 2011. Immune response and pathophysiological features of Klebsiella pneumoniae liver abscesses in an animal model. Lab Invest 91(7): 1029-1039.
    連結:
  12. Geerlings S. E., and Hoepelman A. I. 1999. Immune dysfunction in patients with diabetes mellitus (DM). FEMS Microbiol Lett 26(3-4):259-65.
    連結:
  13. Guenounou M., Smets P., and Agneray J. 1984. Activation of murine B lymphocytes by RU 41740, a glycoprotein extract from Klebsiella pneumoniae. C R Acad Sci III 298(6):135-8.
    連結:
  14. Jiang, P., Li, J., Han, F., Duan, G., Lu, X., Gu, Y., and Yu, W. 2011. Antibiofilm activity of an exopolysaccharide from marine bacterium vibrio sp. QY101. PLoS One 6(4):e18514.
    連結:
  15. Johnson, J.G., Murphy, C.N., Sippy, J., Johnson, T.J., and Clegg, S. 2011. Type 3 fimbriae and biofilm formation are regulated by the transcriptional regulators MrkHI in Klebsiella pneumonia. J Bacteriol 193(14):3453-3460.
    連結:
  16. Klemm P., and Schembri M.A. 2000. Bacterial adhesins: function and structure. Int J Med Microbiol 290(1):27-35.
    連結:
  17. Ko, W.-C., Paterson, D.L., Sagnimeni, A.J., Hansen, D.S., Gottberg, A.V., Mohapatra, S., Casellas, J.M., Goossens, H., Mulazimoglu, L., and Trenholme, G., et al. 2002. Community-Acquired Klebsiella pneumoniae Bacteremia: Global Differences in Clinical Patterns. Emerg Infect Dis 8(2):160-166.
    連結:
  18. J.G. Larkin, B.M. Frier and J.T. Ireland. 1985. Diabetes mellitus and infection. Postgrad Med J 61(713):233-237.
    連結:
  19. Ong C. L., Beatson S. A., Totsika M., Forestier C., McEwan A. G., Schembri M. A. 2010. Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species. BMC Microbiol 24;10:183.
    連結:
  20. Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M., and Galanos, C., et al. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282(5396): 2085-2088.
    連結:
  21. Rittig, M.G., Kaufmann, A., Robins, A., Shaw, B., Sprenger, H., Gemsa, D., Foulongne, V., Rouot, B., and Dornand, J. 2003. Smooth and rough lipopolysaccharide phenotypes of Brucella induce different intracellular trafficking and cytokine/chemokine release in human monocytes. Journal of leukocyte biology 74, 1045-1055. J Leukoc Biol 74(6): 1045-1055.
    連結:
  22. Rukavina T., Ticac B., and Vasiljev V. 2006. IL-10 in antilipopolysaccharide immunity against systemic Klebsiella infections. Mediators Inflamm 2006(6):69431.
    連結:
  23. Schroll C., Barken K. B., Krogfelt K. A., Struve C. 2010. Role of type 1 and type 3 fimbriae in klebsiella pneumoniae biofilm formation. BMC Microbiol 23;10:179.
    連結:
  24. Shankar-Sinha, S., Valencia, G.A., Janes, B.K., Rosenberg, J.K., Whitfield, C., Bender, R.A., Standiford, T.J., and Younger, J.G. 2004. The Klebsiella pneumoniae O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia. Infect Immun 72(3): 1423-1430.
    連結:
  25. Sharma, S., Mohan, H., Sharma, S., and Chhibber, S. 2011. A comparative study of induction of pneumoniae in mice with planktonic and biofilm cells of Klebsiella pneumoniae. Microbiol Immunol 55(5):295-303.
    連結:
  26. Skyberg, J.A., Siek, K.E., Doetkott, C., and Nolan, L.K. 2007. Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny. J Appl Microbiol 102(2):548-554.
    連結:
  27. Stahlhut, S.G., Struve, C., Krogfelt, K.A., and Reisner, A. 2012. Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae. FEMS Immunol Med Microbiol 65(2):350-359.
    連結:
  28. Struve, C., Bojer, M., and Krogfelt K.A. 2008. Characterization of Klebsiella pneumoniae type 1 fimbriae by detection of phase variation during colonization and infection and impact on virulence. Infect Immun 76(9):4055-4065.
    連結:
  29. Takeda K., Kaisho T., and Akira S. 2003. Toll-like receptors. Annu Rev Immunol 21:335-376.
    連結:
  30. Thurlow, L.R., Hanke, M.L., Fritz, T., Angle, A., Aldrich, A., Williams, S.H., Engebretsen, I.L., Bayles, K.W., Horswill, A.R., and Kielian, T. 2011. Staphylococcus aureus biofilms prevent macrophage phagocytosis and attenuate inflammation in vivo. J Immunol 186(11):6585-6596.
    連結:
  31. Tsai, F. C., Huang, Y. T., Chang, L. Y., and Wang, J. T. 2008. Pyogenic Liver Abscess as Endemic Disease, Taiwan. Emerg Infect Dis 14(10):1592-1600.
    連結:
  32. Wang, J. H., Liu, Y. C., Lee, S. S., Yen, M. Y., Chen, Y. S., Wang, J. H., Wann, S. R., and Lin, H.-H. 1998. Primary liver abscess due to Klebsiella pneumoniae in Taiwan. Clin Infect Dis 26(6):1434-1438.
    連結:
  33. Wu, M. C., Lin, T. L., Hsieh, P. F., Yang, H. C., and Wang, J. T. 2011. Isolation of Genes Involved in Biofilm Formation of a Klebsiella pneumoniae Strain Causing Pyogenic Liver Abscess. PLoS One 6(8):e23500.
    連結:
  34. Wood C. D., and Moller G. 1984. Influence of RU 41.740, a glycoprotein extract from Klebsiella pneumonia, on the murine immune syatem. I. T-independent Polyclonal B Cell Activation. J Immunol 132(2):616-21.
    連結:
  35. Wu, H. S., Wang, F. D., Tseng, C. P., Wu, T. H., Lin, Y. T., and Fung, C. P. 2012. Characteristics of healthcare-associated and community-acquired Klebsiella pneumoniae bacteremia in Taiwan. J Infect 64(2): 162-168.
    連結:
  36. Athamna, A., Ofek, I., Keisari, Y., Markowitz, S., Dutton, G.G.S., and Sharon, N. 1991. Lectinophagocytosis of Encapsulated Klebsiella pneumoniae Mediated by Surface Lectins of Guinea Pig Alveolar Macrophages and Human Monocyte-Derived Macrophages. Infect Immun 59(5):1673-1682.
  37. Di Martino P., Bertin Y., Girardeau J.P., Livrelli V., Joly B., and Darfeuile-Michaud A. 1995. Molecular characterization and adhesive properties of CF29K, an adhesin of Klebsiella pneumoniae strainsinvolved in nosocomial infections. Infect Immun 63(11):4336-4344.
  38. Di Martino P., Livrelli V., Sirot D., Joly B., and Darfeuile-Michaud A. 1996. A new fimbrial antigen harbored by CAZ-5/SHV-4-producing Klebsiella pneumoniae strains involved in nosocomial infections. Infect Immun 64(6):2266-2273.
  39. Merino, S., Camprubi, S., Alberti, S., Benedi, V.-J., and Tomas, and J.M. 1992. Mechanisms of Klebsiella pneumoniae Resistance to Complement-Mediated Killing. Infect Immun 60(6):2529-2535.
  40. Mizuta, K., Ohta, M., Mori, M., Hasegawa, T., Nakashima, I., and Kato, N. 1983. Virulence for Mice of Klebsiella Strains Belonging to the 01 Group: Relationship to Their Capsular (K) Types. Infect Immun 40(1):56-61.
  41. Regue, M., Hita, B., Pique, N., Izquierdo, L., Merino, S., Fresno, S., Benedi, V.J., and Tomas, J.M. 2003. A Gene, uge, Is Essential for Klebsiella pneumoniae Virulence. Infection and immunity 72, 54-61. Infect Immun 72(1): 54-61.
  42. Stover, A.G., Correia, J.D.S., Evans, J.T., Cluff, C.W., Elliott, M.W., Jeffery, E.W., Johnson, D.A., Lacy, M.J., Baldridge, J.R., and Probst, P., et al. 2003. Structure-Activity Relationship of Synthetic Toll-like Receptor 4 Agonists. J Biol Chem 279(6):4440-4449.