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系統識別號 U0026-0812200914083182
論文名稱(中文) 鰻魚致病性創傷弧菌共同的毒力質體之鑑識與特性分析
論文名稱(英文) Identification and characterization of a common virulence plasmid in eel-pathogenic Vibrio vulnificus
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 96
學期 2
出版年 97
研究生(中文) 李俊德
研究生(英文) Chung-Te Lee
電子信箱 s5890134@ccmail.ncku.edu.tw
學號 s5890134
學位類別 博士
語文別 英文
論文頁數 100頁
口試委員 口試委員-張晃猷
口試委員-李國誥
口試委員-蔡世峰
指導教授-何漣漪
召集委員-楊惠郎
口試委員-吳俊忠
口試委員-林尊湄
口試委員-鄧景浩
中文關鍵字 創傷弧菌  鰻魚  毒力質體 
英文關鍵字 eels  virulence plasmid  Vibrio vulnificus 
學科別分類
中文摘要 創傷弧菌是一普遍棲居於海水環境的革蘭氏陰性細菌,對人類及水生動物,如鰻魚,具有致病力。創傷弧菌菌株可分成三種生物型,而其中那些對鰻魚具有毒力的菌株屬於生物二型 (biotype 2)。本研究最初目的是欲探討生物二型菌株對鰻魚的毒力因子及其致病機制,之後根據研究的結果發現創傷弧菌的毒力質體,並對其進行鑑識與分析。我先以Suppression Subtractive Hybridization技術鑑別生物一、二型基因體序列的差異,得到三段具有生物二型專一性的DNA序列,這些序列均位於質體上。同時,根據文獻的記載,所有的生物二型菌株都至少帶有一個高分子量的質體,顯示質體似乎與細菌對鰻魚的毒力有關。為了釐清生物二型菌株的質體是否與該型菌株之毒力有關,進一步將兩生物二型菌株中總共三個質體,分別為菌株CECT4602所含的兩個質體pC4602-1 (56,628 bp)及pC4602-2 (66,946 bp),和菌株CECT4999所含的pR99 (68,446 bp),完成其DNA定序並加上基因註解。序列比對的結果顯示,pC4602-2與pR99有92%的DNA序列相同,而且皆帶有一個RTX (repeats-in-toxin) 毒素基因組;pC4602-1則與一個生物一型株中的self-transmissible質體pYJ016有81%的DNA序列相同,並且帶有一組與質體接合傳送有關的基因組。將CECT4999的質體pR99移除,會導致該菌對鰻魚的毒力及抗鰻魚血清殺菌作用的能力大幅降低,但不影響其對小鼠的毒力或抗人類血清殺菌作用的能力。研究中亦發現質體pC4602-1具有self-transmissible的能力,而且在pC4602-1存在下,質體pC4602-2及pR99可利用conjugation的方式傳送入CECT4999的無質體菌株,但此兩質體卻不易傳送至生物一型菌株。質體已移除的CECT4999菌株得到pC4602-2後,其對鰻魚的毒力可恢復,但若只得到pC4602-1則無法恢復毒力。由此可知,pR99與pC4602-2皆為毒力質體。進一步探討一個同時存在於pR99與pC4602-2但功能未知的基因,發現這個基因是細菌抵抗鰻魚血清的殺菌作用與對鰻魚的毒力所必需,但單獨這個基因並不足以達成這兩種性質。此外,研究結果證實CECT4999與CECT4602的染色體上存在另一組具有功能的RTX基因組,而pR99上所encode的RTX toxin對細菌的毒力不是必要的。研究結果也提供了pC4602-1及pC4602-2可能藉由彼此相同的序列以形成一cointegrate的證據。最後,分析其它六株不同來源生物二型菌株的結果發現這些菌株皆帶有毒力質體,而其中四株亦同時帶有與pC4602-1類似的質體。本研究不僅證實創傷弧菌生物二型菌株共同具有一個與該型菌株對鰻魚的毒力相關的質體,也發現此毒力質體可在此型菌株中所發現到另一個能自我傳播的質體的幫忙下,在生物二型菌株間藉接合作用傳遞。
英文摘要 Vibrio vulnificus, a Gram-negative bacterial species ubiquitous in estuarine environments, is pathogenic for humans and aquatic animals such as eels. Strains of V. vulnificus are divided into three biotypes, and those virulent for eels are classified as biotype 2 (BT2). The primary reseach objective was to explore the virulence factors and mechanism of BT2 strains in eels, and our data lead to the identification of a common virulence plasmid of the BT2 strains. Suppression Subtractive Hybridization (SSH) was first conducted to identify the BT2-specific DNA sequences. Three DNA sequences thus identified were detected in all the tested BT2, but not those of other biotypes, strains and they were all located in a common plasmid. This is consistent with a previous finding of a common plasmid of high molecular weight in all the BT2 strains examined. To investigate the role of the plasmid in virulence, the DNA sequences of three BT2 plasmids, pR99 (68,446 bp) in strain CECT4999 as well as pC4602-1 (56,628 bp) and pC4602-2 (66,946 bp) in strain CECT4602, were determined. Plasmids pC4602-2 and pR99 shared 92% sequence identity and contained a gene cluster that encodes the RTX (repeats-in-toxin) toxin. Plasmids pC4602-1 showed 81% sequence identity with a self-transmissible plasmid, pYJ016, in a biotype 1 strain, and contained a gene cluster involved in conjugative gene transfer. Curing of pR99 from strain CECT4999 resulted in loss of resistance to eel serum and virulence for eels, but had no effect on the virulence for mice, an animal model, and resistance to human serum. Plasmids pC4602-2 and pR99 could be transferred to the plasmid-cured strain by conjugation in the presence of pC4602-1 and acquisition of pC4602-2 restored the virulence of the cured strain for eels. Therefore, both pR99 and pC4602-2 were virulence plasmids for eels, but not mice. A gene in pR99, which encoded a novel protein and had an equivalent in pC4602-2, was further shown to be essential, but not sufficient, for the resistance to eel serum and virulence for eels. On the other hand, a functional copy of rtx gene cluster almost identical to that in the plasmid was found in the chromosomes of the BT2 strains, and the copy in pR99 was shown to be dispensable for virulence in eels. There was evidence showing that pC4602-2 may form a cointegrate with pC4602-1 probably via a recombination between the identical sequences present in both plasmids. An investigation of six other biotype 2 strains for the presence of various plasmid markers revealed that they all harbored the virulence plasmid and four of them possessed the conjugal plasmid in addition. In conclusion, this study demonstrated that the virulence of BT2 strains for eels is mediated by a common virulence plasmid, and this plasmid can be disseminated between the BT2 strains by conjugation with the aid of a conjugal plasmid present in some BT2 strains.
論文目次 Text contents
中文摘要 i
Abstract iii
Acknowledgement v
Text contents vi
Table contents ix
Figure contents x
Abbreviation xii
Introduction 1
Materials and methods
1. Bacterial strains, plasmids, and primers used in this study 9
1-1. Bacterial strains 9
1-2. Plasmids 10
1-3. Primers 10

2. DNA extraction and manipulation
2-1. Genomic DNA extraction 10
2-2. Plasmid DNA extraction 11
2-3. Restriction enzyme digestion 11
2-4. DNA ligation 12

3. Suppression Subtractive Hybridization (SSH) 12
4. Cloning of SSH products 12
5. Transformation
5-1. Preparation of the competent cells for heat-shock transformation 13
5-2. Preparation of the competent cells for electroporation 13
5-3. Heat-shock transformation 13
5-4. Electroporation 14

6. Nucleic acid hybridization
6-1. Southern hybridization with Isotope-labeled probes 14
6-2. Southern hybridization with flourescein-labeled probes 15
6-3. Colony hybridization 15
6-4. Dot blot hybridization 16

7. Polymerase chain reaction (PCR)
7-1. PCR to check the specificity of sequences in the subtractive clones 16
7-2. PCR to detect the cointegrate of pC4602-1 and pC4602-2 16

8. DNA sequence determination and analysis 17
9. Isolation of the mutants
9-1. Isolation of CECT4999ΔmazF 18
9-2. Isolation of CECT4999Δvep07 18
9-3. Isolation of CECT4999ΔrtxA 18

10. Cloning of vep07 19
11. Reverse-transcription PCR 19
12. Isolation of the plasmid-cured strain 19
13. Bacterial growth curve 20
14. Serum resistance assay and growth in blood 20
15. Virulence assays
15-1. In eels 20
15-2. In mice 21


16. Bacterial Conjugation 21
17. Ornithine decarboxylase (ODC) test 22
18. Plasmid stability 22
19. Cell culture 22
20. Cytotoxicity assay 22

Results 24
Identification of BT2-specific DNA sequences by SSH 24
Analysis and characterization of the BT2-specific sequences 24
Localization of the BT2-specific sequences in bacterial genome 25
Sequences and features of BT2 plasmid DNA 26
Involvement of pR99 in virulence for eels 27
Association of the gene, vep07, in pR99 with the virulence for eels 28
Role of RTX toxin in BT2 strains 29
Transfer of pC4602-1 and pC4602-2 from strain CECT4602 to BT1 and BT2 strains by conjugation 31

Transfer of pR99 in the presence of pC4602-1 32
Formation of a cointegrate between pC4602-1 and pC4602-2 32
Association of plasmids pC4602-1 and pC4602-2 with virulence for eels 33
Distribution of seq51, traH, mazF, ID1 and ID2 in the plasmids of various BT2 strains 34

Discussion 35
References 42
自述 99

Table contents
1. V. vulnificus strains used to examine the specificity of DNA sequences identified by SSH 53
2. Recombinant or mutant strains of E. coli and V. vulnificus constructed in this study 55
3. Plasmids used in this study 57
4. Primers used in this study 58
5. Features of the BT2-specific DNA sequences 61
6. Summary of the PCR results of V. vulnificus strains tested with primer pairs derived from various BT2-specific DNA sequences 62
7. Similar matches to the ORFs of pC4602-1 in the database 63
8. Similar matches to the ORFs of pC4602-2 in the database 67
9. Similar matches to the ORFs of pR99 in the database 70
10. Association of pR99 with bacterial virulence in eels and mice, and growth in eel and human serum and whole blood 74
11. Association of vep07 with bacterial virulence in eels and growth in eel serum and whole blood 75
12. The frequencies of transferring pC4602-1 and pC4602-2 from strain CECT4602 to CT218 and BT 1 strains by conjugation 76
13. Association of pC4602-1 and pC4602-2 with bacterial virulence in eels and growth in eel serum 77

Figure contents
1. Schematic diagram of suppression subtractive subtraction 78
2. Agarose gel electrophoresis of the products from SSH 79
3. Southern blot hybridization to examine the specificity of SSH products 80
4. Dot blot hybridization to identify the identical subtractive clones 81
5. Southern hybridization to examine the specificity of a cloned sequence 82
6. Southern hybridization of plasmids of various BT2 strains probed with the BT2-specific sequences 83
7. The main features of the plasmids pC4602-1 and pC4602-2 in BT2 strain CECT4602 84
8. The main features of the plasmid, pR99, in V. vulnificus strain CECT4999 85
9. (A) The organization of tra regions of F plasmid, pYJ016 and pC4602-1. (B) A comparison of the nucleotide sequences of pC4602-1 and pYJ016 86
10. Transcriptional levels of mazF in various strains analyzed by RT-PCR 87
11. PCR analysis to confirm a deletion in mazF and an insertion of cat in vep07 of pR99 in CECT4999 88
12. Confirmation of the plasmid-cured strain, CT218, by PCR, plasmid profile analysis and Southern hybridization 89
13. Growth of CECT4999, CT218, and CT188 in LB medium 90
14. Southern hybridization to confirm the deletion (225 bp) in vep07 91
15. Comparison of the rtxA nucleotide sequences in V. cholerae strain N16961 (rtxAVc), V. vulnificus BT1 strain YJ016 (rtxAVvbt1) and pC4602-2 (rtxAVvbt2) 92
16. Analyses of the rtx gene clusters in the chromosomes of strains CECT4999 and CECT4602 93
17. Southern hybridization to confirm the ΔrtxA mutants, CT284 and CT285 94
18. Cytotoxicity of various strains with HEp-2 cells 95
19. The profiles of uncut and BglII-restricted plasmids of various transconjugants 96
20. Formation of cointegrates of plasmids pC4602-1 and pC4602-2 97
21. The distribution of genes and regions involved in virulence for eels, plasmid transfer, and plasmid stability in the plasmids of various BT2 strains 98
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