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研究生:陳治忠
論文名稱:創傷弧菌中多白胺酸重複基因lrv之研究
論文名稱(外文):Characterization of Leucine Rich Repeat Containing Gene lrv of Vibrio vulnificus
指導教授:張晃猷
指導教授(外文):Hwan-You Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
中文關鍵詞:創傷弧菌多白胺酸重複基因蛋白質反應區域同源染色體互換細胞分散
外文關鍵詞:Vibrio vulnificusLeucine Rich RepeatProtein-protein interaction domainIronHomologous recombinationCell dispersion
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中文摘要
創傷弧菌為一嗜鹽性革蘭氏陰性桿菌, 對人類造成嚴重的傷口感染和致命性敗血症。在創傷弧菌的基因體被完整解出及註解後,我們在其基因體中找到一段包含多白氨酸重複序列基因。多白氨酸重複序列是一種常見的蛋白質反應區域,在某些微生物中也找到包含此重複序列的基因,例如Shigella flexneri中的IpaH家族,Yersinia specie中的YopM ,以及Listeria monocytogenes中的internalin家族,以上基因皆和細菌的致病性有關。為了探討多白氨酸重複序列基因在創傷弧菌中可能扮演的功能,我選殖此段基因並在大腸桿菌中重組以生產重組蛋白及製造抗體。我發現多白氨酸重複基因是一個外膜蛋白並且在生物一型和生物二型的創傷弧菌中皆會產生。此外,鐵對創傷弧菌而言是一非常重要的毒力調控因子,我發現多白氨酸重複蛋白在缺乏鐵的環境下會大量產生。利用同源染色體互換的技術,我製造了缺乏白氨酸重複蛋白的突變株,但在以腹腔注射為感染途徑的小鼠毒力測試中,突變株並不會降低對小鼠的毒力。在細胞感染實驗方面,我發現當野生株感染細胞株時,細胞會出現分散的型態,而當突變株感染細胞株時,細胞分散的型態已不明顯。我認為創傷弧菌中多白氨酸重複蛋白可能影響細胞株之間的連結,以幫助創傷弧菌具有侵略性的感染。

Abstract
Vibrio vulnificus is a halophilic, Gram-negative, curved rod-shaped bacterium. This bacterium has the ability to cause serious wound infections and often-fatal septicemia in human. After the genome of V. vulnificus has been completely sequenced and annotated, we have identified a gene named lrv that contains Leucine-rich repeats (LRRs) domain, a common property involving in protein-protein interaction. Several bacterial genes encoding an LRR protein have been reported, which include the IpaH family in Shigella fexneri, YopM in Yersinia species, and internalin family in Listeria monocytogenes, and are normally associated with pathogenesis. In order to explore the possible function of lrv in V. vulnificus, I cloned and expressed the gene, and produced recombinant Lrv and an anti-Lrv antibody. I found that Lrv is an outer membrane protein and expressed in both biotypes 1 and 2 of V. vulnificus. Lrv expression is up regulated in iron-deficient condition. I have constructed a lrv deletion mutant by the allelic exchange technique, and found that the LD 50 of the mutant did not change in animal intraperitoneal (i.p.) model of infection. The mutant strain appeared to be less able to cause cell dispersion compared to the parental strain. In conclusion, our data suggest that Lrv may disturb the cell-cell junction and facilitate the invasive infection of V. vulnificus.

CONTENTS
ABSTRACT (CHINESE)……………………………………………………… i
ABSTRACT……………………………………………………………………… ii
ACKNOWLEDGMENT…………………………………………………………iii
CONTENTS……………………………………………………………………….iv
TABLE CONTENTS…………………………………………………………….vi
FIGURE CONTENTS…………………………………………………………..vii
INTRODUCTION
1.The organism…………………………………………………………………. 1
2. Biotypes……………………………………………………………………… 1
3. Host susceptibility…………………………………………………………… 2
4. Clinical symptoms and treatment……………………………………………. 2
5. Virulence of V. vulnificus…………………………………………………………… 3
5.1. Polysaccharide capsule and LPS……………………………………….. 3
5.2. Pili…………………………………………………………………………3
5.3. Hemolysins…………………………………………………………………4
5.4. Metalloprotease…………………………………………………………….4
5.5 Enterotoxin………………………………………………………………….5
5.6 Other Extracellular Proteins……………………………………………….. 5
5.7 Iron………………………………………………………………………….5
6. This study……………………………………………………………………….6
MATERIAL AND METHODS
Bioinformatic analysis……………………………………………………………8
Bacterial strains, mammalian cell lines and plasmids …………………………...8
Media, cultivation and storage of bacteria and cell lines……………………….. 8
RT-PCR……………………………………………………………………… ….9
Genomic DNA preparation and PCR……………………………………………9
Construction of expression plasmids……………………………………………10
Protein expression, purification and refolding…………………………………..10
Polyclonal antibody production…………………………………………………10
Sub-cellular fractionation……………………………………………………….11
Western blot analysis……………………………………………………………11
Cell line assay with treatment of recombinant Lrv……………………………..12
Construction of lrv deletion mutant in V. vulnificus YJ016……………………12
Southern hybridization ………………………………………………………13
Determination of bacterial density ……………………………………………13
Cell line infection assay ………………………………………………………14
Virulence assay…………………………………………………………………14
RESULTS……………………………………………………………………….. 15
Features of lrv……………………………………………………………………………15
lrv is expressed in V.vulnificus…………………………………………………….15
Production of Lrv protein in E. coli……………………………………………………16
Generation of a Lrv-specific polyclonal antibody……………………………….16
lrv expression level increased as the bacterium grows…………………………...16
Both biotypes 1 and 2 of V. vulnificus express lrv……………………………….17
lrv expression is associated with iron deficiency………………………………...17
Lrv is an outer membrane protein …………………………………………….17
Recombinant Lrv alters MDCK cell morphology………………………………..18
Isolation of V. vulnificus lrv -deletion mutant……………………………………18
The ability to cause cell dispersion is reduced in the V. vulnificus lrv deletion
mutant……………………………………………………………………………19
The virulence of V. vulnificus lrv deletion mutant is not reduced……………….19
DISCUSSION…………………………………………………………………. 20
REFERENCES………………………………………………………………… 23
TABLES AND FIGURES ………………………………………………………28

Reference
1. Amaro, C., and E. G. Biosca. 1996. Vibrio vulnificus biotype 2, pathogenic for eels, is also an opportunistic pathogen for humans. Appl Environ Microbiol. 62:1454-7.
2. Amaro, C., E. G. Biosca, B. Fouz, A. E. Toranzo, and E. Garay. 1994. Role of iron, capsule, and toxins in the pathogenicity of Vibrio vulnificus biotype 2 for mice. Infect Immun. 62:759-63.
3. Birchmeier, C., and E. Gherardi. 1998. Developmental roles of HGF/SF and its receptor, the c-Met tyrosine kinase. Trends Cell Biol. 8:404-10.
4. Brennt, C. E., A. C. Wright, S. K. Dutta, and J. G. Morris, Jr. 1991. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J Infect Dis. 164:1030-2.
5. Bullen, J. J., P. B. Spalding, C. G. Ward, and J. M. Gutteridge. 1991. Hemochromatosis, iron and septicemia caused by Vibrio vulnificus. Arch Intern Med. 151:1606-9.
6. Domann, E., S. Zechel, A. Lingnau, T. Hain, A. Darji, T. Nichterlein, J. Wehland, and T. Chakraborty. 1997. Identification and characterization of a novel PrfA-regulated gene in Listeria monocytogenes whose product, IrpA, is highly homologous to internalin proteins, which contain leucine-rich repeats. Infect Immun. 65:101-9.
7. Dramsi, S., P. Dehoux, M. Lebrun, P. L. Goossens, and P. Cossart. 1997. Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD. Infect Immun. 65:1615-25.
8. Engelbrecht, F., S. K. Chun, C. Ochs, J. Hess, F. Lottspeich, W. Goebel, and Z. Sokolovic. 1996. A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins. Mol Microbiol. 21:823-37.
9. Gaillard, J. L., P. Berche, C. Frehel, E. Gouin, and P. Cossart. 1991. Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from gram-positive cocci. Cell. 65:1127-41.
10. Gander, R. M., and M. T. LaRocco. 1989. Detection of piluslike structures on clinical and environmental isolates of Vibrio vulnificus. J Clin Microbiol. 27:1015-21.
11. Gray, L. D., and A. S. Kreger. 1985. Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus. Infect Immun. 48:62-72.
12. Helms, S. D., J. D. Oliver, and J. C. Travis. 1984. Role of heme compounds and haptoglobin in Vibrio vulnificus pathogenicity. Infect Immun. 45:345-9.
13. Klontz, K. C., S. Lieb, M. Schreiber, H. T. Janowski, L. M. Baldy, and R. A. Gunn. 1988. Syndromes of Vibrio vulnificus infections. Clinical and epidemiologic features in Florida cases, 1981-1987. Ann Intern Med. 109:318-23.
14. Kobe, B., and J. Deisenhofer. 1994. The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci. 19:415-21.
15. Kobe, B., and A. V. Kajava. 2001. The leucine-rich repeat as a protein recognition motif. Curr Opin Struct Biol. 11:725-32.
16. Koenig, K. L., J. Mueller, and T. Rose. 1991. Vibrio vulnificus. Hazard on the half shell. West J Med. 155:400-3.
17. Kothary, M. H., and A. S. Kreger. 1987. Purification and characterization of an elastolytic protease of Vibrio vulnificus. J Gen Microbiol. 133 ( Pt 7):1783-91.
18. Kraffert, C. A., and D. J. Hogan. 1992. Vibrio vulnificus infection and iron overload. J Am Acad Dermatol. 26:140.
19. Leung, K. Y., B. S. Reisner, and S. C. Straley. 1990. YopM inhibits platelet aggregation and is necessary for virulence of Yersinia pestis in mice. Infect Immun. 58:3262-71.
20. Leung, K. Y., and S. C. Straley. 1989. The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb alpha. J Bacteriol. 171:4623-32.
21. Litwin, C. M., and S. B. Calderwood. 1993. Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J Bacteriol. 175:706-15.
22. Mengaud, J., H. Ohayon, P. Gounon, R. M. Mege, and P. Cossart. 1996. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell. 84:923-32.
23. Miyoshi, N., C. Shimizu, S. Miyoshi, and S. Shinoda. 1987. Purification and characterization of Vibrio vulnificus protease. Microbiol Immunol. 31:13-25.
24. Moreno, M. L., and M. Landgraf. 1998. Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood. J Appl Microbiol. 84:747-51.
25. Powell, J. L., A. C. Wright, S. S. Wasserman, D. M. Hone, and J. G. Morris, Jr. 1997. Release of tumor necrosis factor alpha in response to Vibrio vulnificus capsular polysaccharide in in vivo and in vitro models. Infect Immun. 65:3713-8.
26. Raffelsbauer, D., A. Bubert, F. Engelbrecht, J. Scheinpflug, A. Simm, J. Hess, S. H. Kaufmann, and W. Goebel. 1998. The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice. Mol Gen Genet. 260:144-58.
27. Reed, L. J., and H. Muench. 1938. A simple method of estimating the fifty percent endpoint. AM. J. Hyg. 27:493-497.
28. Rodrigues, D. P., R. V. Ribeiro, and E. Hofer. 1992. Analysis of some virulence factors of Vibrio vulnificus isolated from Rio de Janeiro, Brazil. Epidemiol Infect. 108:463-7.
29. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J Bacteriol. 108:545-52.
30. Shapiro, R. L., S. Altekruse, L. Hutwagner, R. Bishop, R. Hammond, S. Wilson, B. Ray, S. Thompson, R. V. Tauxe, and P. M. Griffin. 1998. The role of Gulf Coast oysters harvested in warmer months in Vibrio vulnificus infections in the United States, 1988-1996. Vibrio Working Group. J Infect Dis. 178:752-9.
31. Shen, Y., M. Naujokas, M. Park, and K. Ireton. 2000. InIB-dependent internalization of Listeria is mediated by the Met receptor tyrosine kinase. Cell. 103:501-10.
32. Simpson, L. M., V. K. White, S. F. Zane, and J. D. Oliver. 1987. Correlation between virulence and colony morphology in Vibrio vulnificus. Infect Immun. 55:269-72.
33. Smith, G. C., and J. R. Merkel. 1982. Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness. Infect Immun. 35:1155-6.
34. Stelma, G. N., Jr., A. L. Reyes, J. T. Peeler, C. H. Johnson, and P. L. Spaulding. 1992. Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus. Appl Environ Microbiol. 58:2776-82.
35. Tison, D. L., M. Nishibuchi, J. D. Greenwood, and R. J. Seidler. 1982. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol. 44:640-6.
36. Veenstra, J., P. J. Rietra, J. Goudswaard, J. A. Kaan, P. H. van Keulen, and C. P. Stoutenbeek. 1993. Extra-intestinal infections caused by Vibrio spp. in The Netherlands. Ned Tijdschr Geneeskd. 137:654-7.
37. Venkatesan, M. M., J. M. Buysse, and A. B. Hartman. 1991. Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Mol Microbiol. 5:2435-45.
38. Wright, A. C., L. M. Simpson, and J. D. Oliver. 1981. Role of iron in the pathogenesis of Vibrio vulnificus infections. Infect Immun. 34:503-7.
39. Wright, A. C., L. M. Simpson, J. D. Oliver, and J. G. Morris, Jr. 1990. Phenotypic evaluation of acapsular transposon mutants of Vibrio vulnificus. Infect Immun. 58:1769-73.
40. Yoshida, S., M. Ogawa, and Y. Mizuguchi. 1985. Relation of capsular materials and colony opacity to virulence of Vibrio vulnificus. Infect Immun. 47:446-51.

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