臺灣博碩士論文加值系統

　　創傷弧菌為一伺機感染性病原菌，主要生存於海水內，會經由傷口或胃腸道而造成感染。目前推測創傷弧菌的毒力因子主要有莢膜、胞外產物、細菌競爭宿主體內鐵的能力等，但關於創傷弧菌的致病機制仍不清楚。近年來，已有許多研究顯示quorum-sensing system會調控某些致病菌的毒力，如霍亂弧菌、綠膿桿菌及金黃色葡萄球菌等。創傷弧菌的基因體序列已於2002年全部定序完成，且經由annotation的結果得知參與AI-2 quorum-sensing system的基因都可在創傷弧菌中被比對出來。本研究的目的在於了解創傷弧菌於感染小鼠的過程中，其毒力因子的表現是否受到quorum-sensing system的調控。藉由分離及分析此系統調控子LuxO的兩種表現型，一為luxO缺失（LD）突變，另一為LuxO持續活化的luxOD47E突變，我們發現LuxO不但會負調控創傷弧菌蛋白酶的表現同時也會正調控細胞溶解毒素的表現。在測定不同luxO突變株以皮下感染模式對實驗小鼠的半致死劑量的實驗中，我們觀察到LD突變株的致死力比野生株或luxOD47E突變株下降近100倍，顯示LuxO的突變確實會影響創傷弧菌的毒力。進一步測試luxO突變株對HEp-2細胞的毒性時，發現luxOD47E突變株的細胞毒性與野生株並沒有明顯差異，但LD突變株的細胞毒性已喪失。此外，我們也發現LD突變株由感染部位侵入小鼠循環系統的散播能力遠比野生株或是luxOD47E突變株差，並且觀察各突變株由皮下感染小鼠之上皮組織的病理變化時，亦發現受LD突變株感染後，只有大量吞噬細胞遍布於真皮層及皮下層，而無細菌的聚集。但受luxOD47E突變株感染的結果則與野生株相彷，均可見到大量吞噬細胞及細菌聚集於感染部位的真皮層及皮下層。另一方面，分析各突變株所產生siderophore的表現量，在小鼠全血內的生長情形，及對人體血清抗性的實驗，其結果均顯現各突變株的表現與野生株並無明顯差異。因此，根據以上實驗結果，我們推測LuxO調控創傷弧菌的毒力表現可能是藉由活化某一細胞毒素，且此毒素對於此菌抵抗免疫細胞的攻擊或是侵入小鼠循環系統的能力扮演了相當重要的角色。

　　Vibrio vulnificus, a halophilic gram-negative marine bacterium, is an opportunistic pathogen causing septicemia and wound infection in humans. A few virulence factors, such as the capsule, a few extracellular products, and iron-acquisition ability, have been proposed; however, the pathogenesis mechanism of this organism remains unclear. It has been shown previously that the quorum-sensing system can regulate the virulence of pathogens, such as V. cholerae, Pseudomonas aeruginosa, and Staphylococcus aureus. In the whole genome sequence of V. vulnificus completed in 2002, most genes required for AI-2 quorum-sensing signaling were identified. To understand whether the quorum-sensing system is involved in the regulation of V. vulnificus, a mutant (LD mutant) deficient in LuxO, the quorum-sensing regulator, and a luxOD47E mutant expressing constitutively active LuxO were isolated and characterized. We found that LuxO regulates not only the extracellular protease activity negatively but also the cytolysin activity positively. The virulence of LD mutant in mice infected by subcutaneous injection was 100-fold less than that of the wild-type strain or the luxOD47E mutant, suggesting that quorum-sensing system may be involved in regulating the virulence of V. vulnificus. The cytotoxicity to HEp-2 cells was not affected in the luxOD47E mutant, but has diminished in the LD mutant. Furthermore, the spread of bacteria from subcutaneous tissue to bloodstream was remarkably slower in mice infected with the LD mutant than those infected with the wild-type strain or luxOD47E mutant. In the histological examination, we found abundant infiltrating cells but few bacteria in the infection site with the LD mutant. However, a large number of infiltrating cells and bacteria were found clustered together with the wild-type strain and the luxOD47E mutant. On the other hand, the amount of siderophore produced, growth in murine blood, and resistance to serum bactericidal effect were not affected in either the LD or luxOD47E mutant. In conclusion, these results suggest that LuxO may be involved in regulating the virulence of V. vulnificus, probably via activating the expression of an unknown cytotoxin important for escaping the attack from the phagocytes and/or invasion of this organism to the bloodstream, where the organism multiplies and causes septic shock and death.

中文摘要 i
英文摘要 ii
致謝 iii
目錄 iv
表目錄 viii
圖目錄 ix
符號及縮寫 x
緒論 1
材料與方法 8
（1） 實驗菌株、質體及實驗動物 8
（2） 實驗菌種培養及其保存 8
（3） 實驗方法 8
核酸技術
1. 商業化套件萃取質體DNA 8
2. 小量純化質體DNA 9
3. 大量純化質體DNA 9
4. 創傷弧菌染色體DNA萃取 10
5. 聚合酶連鎖反應 11
6. 限制酶切割DNA 11
7. DNA電泳分析 11
8. DNA片段之分離與回收 11
9. DNA片段之粘端補齊反應 12
10. DNA片段之去磷酸化反應 12
11. DNA接合反應 13
12. DNA定點突變反應 13
13. 細菌之RNA萃取 13
14. 細菌RNA反轉錄作用及聚合酶連鎖反應 14
雜交試驗
1. DNA探針製備 14
2. 南方雜交法 15
質體轉移方法
1. 熱休克轉形作用 15
2. 電極轉形作用 16
3. 接合生殖作用 16
（4） 創傷弧菌突變株之製備 17
（5） 互補作用 17
（6） 細菌生長曲線之測定 18
（7） 蛋白酶、細胞溶解毒素活性及siderophore表現之測試 18
1. 蛋白酶活性測試 18
2. 細胞溶解毒素活性測試 18
3. Siderophore表現測試 19
（8） 細胞培養及細胞毒性分析 19
1. 細胞培養 19
2. 細胞毒性分析 19
（9）動物實驗 20
1. 細菌對實驗小鼠半致死劑量的測定 20
2. 細菌侵襲小鼠循環系統之能力 20
3. 細菌在小鼠血液中生長能力的測定 20
4. 細菌對人體血清殺菌作用之抗性 20
5. 組織學分析 21
（10）資料分析
1. 創傷弧菌基因資料庫比對 21
2. DNA序列分析 21
結果 23
（1） 創傷弧菌quorum sensing system之鑑識與分析 23
1. 創傷弧菌quorum sensing system BLAST比對之結果 23
（2） 創傷弧菌luxO突變株之分離 24
1. 創傷弧菌�惻uxO基因突變株的分離 24
2. 創傷弧菌LD之補償株（complemented strain）分離 25
3. 創傷弧菌luxOD47E突變株分離 25
（3）創傷弧菌luxO突變株之生長情形 26
（4）創傷弧菌luxO突變株對蛋白酶與細胞溶解毒素表現之調控 26
（5）創傷弧菌luxO突變株在小鼠致病過程中之角色 27
1、luxO突變株對小鼠的致死力 27
2、luxO突變株對上皮細胞毒殺作用之比較 27
3、luxO突變株侵襲小鼠循環系統之能力 28
4、luxO突變株對人類血清殺菌作用之抗性 29
5、luxO突變株對創傷弧菌siderophore表現量的影響 29
6、luxO突變株在小鼠血液中生長的能力 29
7、感染部位的變化 29
討論 31
參考文獻 36
表 40
圖 45

1.Amako, K., K. Okada, and S. Miake. 1984. Evidence for the presence of
a capsule in Vibrio vulnificus. J Gen Microbiol 130 ( Pt 10):2741-3.
2.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.
3.Amaro, C., E. G. Biosca, B. Fouz, E. Alcaide, and C. Esteve. 1995.
Evidence that water transmits Vibrio vulnificus biotype 2 infections
to eels. Appl Environ Microbiol 61:1133-7.
4.Amaro, C., E. G. Biosca, B. Fouz, and E. Garay. 1992. Electrophoretic
analysis of heterogeneous lipopolysaccharides from various strains of
Vibrio vulnificus biotypes 1 and 2 by silver staining and
immunoblotting. Curr Microbiol 25:99-104.
5.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.
6.Bassler, B. L. 2002. Small talk. Cell-to-cell communication in
bacteria. Cell 109:421-4.
7.Bassler, B. L., M. Wright, and M. R. Silverman. 1994. Multiple
signalling systems controlling expression of luminescence in Vibrio
harveyi: sequence and function of genes encoding a second sensory
pathway. Mol Microbiol 13:273-86.
8.Bassler, B. L., M. Wright, and M. R. Silverman. 1994. Sequence and
function of LuxO, a negative regulator of luminescence in Vibrio
harveyi. Mol Microbiol 12:403-12.
9.Biosca, E. G., B. Fouz, E. Alcaide, and C. Amaro. 1996. Siderophore-
mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2.
Appl Environ Microbiol 62:928-35.
10.Bisharat, N., V. Agmon, R. Finkelstein, R. Raz, G. Ben-Dror, L.
Lerner, S. Soboh, R. Colodner, D. N. Cameron, D. L. Wykstra, D. L.
Swerdlow, and J. J. Farmer, 3rd. 1999. Clinical, epidemiological, and
microbiological features of Vibrio vulnificus biogroup 3 causing
outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio
Study Group. Lancet 354:1421-4.
11.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.
12.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.
13.Chan, T. Y., D. P. Chow, K. C. Ng, K. W. Pang, and G. A. McBride.
1994. Vibrio vulnificus septicemia in a patient with liver cirrhosis.
Southeast Asian J Trop Med Public Health 25:215-6.
14.Chen, C. Y., K. M. Wu, Y. C. Chang, C. H. Chang, H. C. Tsai, T. L.
Liao, Y. M. Liu, H. J. Chen, A. B. Shen, J. C. Li, T. L. Su, C. P.
Shao, C. T. Lee, L. I. Hor, and S. F. Tsai. 2003. Comparative genome
analysis of Vibrio vulnificus, a marine pathogen. Genome Res 13:2577-
87.
15.Donnenberg, M. S., and J. B. Kaper. 1991. Construction of an eae
deletion mutant of enteropathogenic Escherichia coli by using a
positive-selection suicide vector. Infect Immun 59:4310-7.
16.Engebrecht, J., and M. Silverman. 1984. Identification of genes and
gene products necessary for bacterial bioluminescence. Proc Natl Acad
Sci U S A 81:4154-8.
17.Freeman, J. A., and B. L. Bassler. 1999. A genetic analysis of the
function of LuxO, a two-component response regulator involved in
quorum sensing in Vibrio harveyi. Mol Microbiol 31:665-77.
18.Freeman, J. A., and B. L. Bassler. 1999. Sequence and function of
LuxU: a two-component phosphorelay protein that regulates quorum
sensing in Vibrio harveyi. J Bacteriol 181:899-906.
19.Freeman, J. A., B. N. Lilley, and B. L. Bassler. 2000. A genetic
analysis of the functions of LuxN: a two-component hybrid sensor
kinase that regulates quorum sensing in Vibrio harveyi. Mol Microbiol
35:139-49.
20.Hanahan, D. 1983. Studies on transformation of Escherichia coli with
plasmids. J Mol Biol 166:557-80.
21.Hayat, U., G. P. Reddy, C. A. Bush, J. A. Johnson, A. C. Wright, and
J. G. Morris, Jr. 1993. Capsular types of Vibrio vulnificus: an
analysis of strains from clinical and environmental sources. J Infect
Dis 168:758-62.
22.Hollis, D. G., R. E. Weaver, C. N. Baker, and C. Thornsberry. 1976.
Halophilic Vibrio species isolated from blood cultures. J Clin
Microbiol 3:425-31.
23.Hor, L. I., Y. K. Chang, C. C. Chang, H. Y. Lei, and J. T. Ou. 2000.
Mechanism of high susceptibility of iron-overloaded mouse to Vibrio
vulnificus infection. Microbiol Immunol 44:871-8.
24.Kreger, A., and D. Lockwood. 1981. Detection of extracellular toxin
(s) produced by Vibrio vulnificus. Infect Immun 33:583-90.
25.Lee, S. E., S. H. Shin, S. Y. Kim, Y. R. Kim, D. H. Shin, S. S.
Chung, Z. H. Lee, J. Y. Lee, K. C. Jeong, S. H. Choi, and J. H. Rhee.
2000. Vibrio vulnificus has the transmembrane transcription activator
ToxRS stimulating the expression of the hemolysin gene vvhA. J
Bacteriol 182:3405-15.
26.Lilley, B. N., and B. L. Bassler. 2000. Regulation of quorum sensing
in Vibrio harveyi by LuxO and sigma-54. Mol Microbiol 36:940-54.
27.Litwin, C. M., T. W. Rayback, and J. Skinner. 1996. Role of catechol
siderophore synthesis in Vibrio vulnificus virulence. Infect Immun
64:2834-8.
28.Manoil, C., and J. Beckwith. 1985. TnphoA: a transposon probe for
protein export signals. Proc Natl Acad Sci U S A 82:8129-33.
29.McKnight, S. L., B. H. Iglewski, and E. C. Pesci. 2000. The
Pseudomonas quinolone signal regulates rhl quorum sensing in
Pseudomonas aeruginosa. J Bacteriol 182:2702-8.
30.Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria.
Annu Rev Microbiol 55:165-99.
31.Miller, M. B., K. Skorupski, D. H. Lenz, R. K. Taylor, and B. L.
Bassler. 2002. Parallel quorum sensing systems converge to regulate
virulence in Vibrio cholerae. Cell 110:303-14.
32.Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and
its use in construction of insertion mutations: osmoregulation of
outer membrane proteins and virulence determinants in Vibrio cholerae
requires toxR. J Bacteriol 170:2575-83.
33.Nealson, K. H., T. Platt, and J. W. Hastings. 1970. Cellular control
of the synthesis and activity of the bacterial luminescent system. J
Bacteriol 104:313-22.
34.O'Neill, K. R., S. H. Jones, and D. J. Grimes. 1992. Seasonal
incidence of Vibrio vulnificus in the Great Bay estuary of New
Hampshire and Maine. Appl Environ Microbiol 58:3257-62.
35.Redd, L. Y., and H. Muench. 1938. A simple method of estimating the
fifty percent endpoints. Am. J. Hyg. 27:493.
36.Reddy, G. P., U. Hayat, C. Abeygunawardana, C. Fox, A. C. Wright, D.
R. Maneval, Jr., C. A. Bush, and J. G. Morris, Jr. 1992. Purification
and determination of the structure of capsular polysaccharide of
Vibrio vulnificus M06-24. J Bacteriol 174:2620-30.
37.Ruby, E. G., and M. J. McFall-Ngai. 1992. A squid that glows in the
night: development of an animal-bacterial mutualism. J Bacteriol
174:4865-70.
38.Schwyn, B., and J. B. Neilands. 1987. Universal chemical assay for
the detection and determination of siderophores. Anal Biochem 160:47-
56.
39.Shao, C. P., and L. I. Hor. 2000. Metalloprotease is not essential
for Vibrio vulnificus virulence in mice. Infect Immun 68:3569-73.
40.Shao, C. P., and L. I. Hor. 2001. Regulation of metalloprotease gene
expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue. J
Bacteriol 183:1369-75.
41.Singh, P. K., A. L. Schaefer, M. R. Parsek, T. O. Moninger, M. J.
Welsh, and E. P. Greenberg. 2000. Quorum-sensing signals indicate
that cystic fibrosis lungs are infected with bacterial biofilms.
Nature 407:762-4.
42.Strom, M. S., and R. N. Paranjpye. 2000. Epidemiology and
pathogenesis of Vibrio vulnificus. Microbes Infect 2:177-88.
43.Subramani, S. 1992. Targeting of proteins into the peroxisomal
matrix. J Membr Biol 125:99-106.
44.Vance, R. E., J. Zhu, and J. J. Mekalanos. 2003. A constitutively
active variant of the quorum-sensing regulator LuxO affects protease
production and biofilm formation in Vibrio cholerae. Infect Immun
71:2571-6.
45.Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13
phage cloning vectors and host strains: nucleotide sequences of the
M13mp18 and pUC19 vectors. Gene 33:103-19.
46.Yarwood, J. M., and P. M. Schlievert. 2003. Quorum sensing in
Staphylococcus infections. J Clin Invest 112:1620-5.
47.Zhu, J., M. B. Miller, R. E. Vance, M. Dziejman, B. L. Bassler, and
J. J. Mekalanos. 2002. Quorum-sensing regulators control virulence
gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A 99:3129-
34.