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論文名稱(外文):The roles of ACD and ERM domains in the functions of Vibrio vulnificus biotype 2 RTX toxin
指導教授(外文):Lien-I Hor
外文關鍵詞:Vibrio vulnificusRTX toxinACD domainERM domaincytotoxicityantiphagocytosis
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創傷弧菌為一革蘭氏陰性菌,通常棲居於淡鹹水交界處,主要分為三種生物型,皆可感染人類,但只有生物二型因具有一毒力質體,而對鰻魚有致病性。此菌產生各種致病因子,其所分泌之RTX毒素為主要因子之一。此毒素可在許多革蘭氏陰性菌中發現,對宿主所造成的影響也各不相同;創傷弧菌之RTX毒素具細胞毒殺性(cytotoxicity)及抗吞噬(antiphagocytosis)功能,如將其剔除,會造成創傷弧菌對小鼠的毒力顯著下降,顯示此毒素在創傷弧菌感染過程中是不可或缺的。我們實驗室先前發現,生物一型及生物二型菌株之RTX毒素所含的domains不盡相同。此外,生物一型菌株只在染色體上帶有一套RTX毒素基因組,但生物二型菌株之毒力質體及染色體上各帶有一套相同的RTX毒素基因組。於本論文研究中,我探討了生物二型RTX毒素之ACD(actin cross-linking domain)及 ERM (ezrin-radixin-moesin) domain於此毒素之功能上的角色。ACD也存在於霍亂弧菌(V. cholerae)之RTX毒素中,且已知能造成宿主細胞的actin產生cross-linking,但並不存在於生物一型RTX毒素中;ERM domain則為此三種RTX毒素所共有,雖與宿主的actin-binding蛋白在胺基酸序列上有相似性,但其在此毒素中的功能未知。我發現將生物二型RTX毒素中的ACD domain剔除後所得到的突變株GG036雖失去了造成actin cross-linking的功能,但仍保有細胞毒殺性及抗吞噬能力,顯示ACD domain並不參與此毒素之細胞毒殺性及抗吞噬功能。另一方面,將ERM domain剔除之突變株GG038則完全喪失細胞毒殺性及抗吞噬能力。但我接著發現GG038同時也失去了ACD的功能,推測剔除ERM domain後RTX毒素失去功能的原因可能是因為RTX毒素失去了其正確蛋白質結構。我進一步分別剔除了ERM domain N端73 a.a.(得突變株GG057)及C端34 a.a.(得突變株GG059)後發現,GG059表現微弱的actin cross-linking的功能,且仍保有約31%之細胞毒殺性及87.7%抗吞噬能力,此結果和我們認為ERM domain與維持RTX毒素蛋白正確構型相關的推測一致。以免疫螢光顯微鏡觀察野生株及各突變株RTX毒素感染宿主細胞後在細胞中的分布及濃度,GG036及GG059與CECT4999螢光訊號強度相當。但GG038及GG057感染後,宿主細胞內RTX蛋白之訊號則明顯較弱,顯示剔除部份或全部之ERM domain的RTX蛋白可能因結構改變而失去與宿主細胞作用的能力。
Vibrio vulnificus, a gram-negative bacterial species inhabiting estuary, is divided into three biotypes (BTs). All BTs may infect humans, but only the BT2 strains are pathogenic for the eels due to the presence of a virulence plasmid. This species produces a variety of virulence factors, with the RTX (repeats in toxin) toxin as one of them. The RTX family members could be found in many gram-negative bacteria, and they exert different effects on the hosts. The V. vulnificus RTX toxin confers cytotoxicity and antiphagocytosis, and a mutant deficient in this toxin exhibits significantly reduced virulence in the mouse, indicating that it is indispensible in pathogenesis. Our laboratory has previously found that the RTX toxins of BT1 and BT2 strains contain dissimilar domains. In addition, unlike the BT1 strains, which contain only one copy of rtx gene cluster in the chromosome, the BT2 strains possess two identical copies, each in the chromosome and virulence plasmid. In this study, I investigated the roles of ACD (actin cross-linking domain) and ERM (ezrin-radizin-moeisin) domains in the functions of BT2 RTX toxin. The ACD domain is also present in the V. cholerae RTX toxin and is known to cause actin cross-linking in the host cell, but it does not exist in the BT1 RTX toxin. The ERM domain is found in all the three RTX toxins, and although it shares sequence homology with the eukaryotic actin-binding proteins, its function remains unclear. I found that a mutant, GG036, deleted of the ACD domain lost the actin cross-linking ability, but was still cytotoxic and resistant to phagocytosis, indicating that this domain is not involved in these two properties. On the other hand, a mutant deleted of the ERM domain (GG038) lost cytotoxicity and the abilities of antiphagocytosis and actin cross-linking. This suggests that the RTX without the ERM domain may not form a functional protein structure. I further isolated mutants that were deleted of 73 residues at the N-terminus (mutant GG057) and 34 residues at the C-terminus (mutant GG059) of ERM domain. It was shown that GG059 retained 31% cytotoxicity, 87.7% antiphagocytosis ability and weak actin cross-linking ability while GG057 was defective in all of these properties. This result is consistent with our speculation that the ERM domain is associated with formation of the functional RTX structure. An examination of the amounts and distribution of RTX in the infected cells by immunofluorescent microscopy revealed that GG036 and GG059 resulted in RTX signals comparable to that caused by the wild-type strain. However, mutants GG038 and GG057 resulted in significantly weaker RTX signals, suggesting that deletion of the entire or part of ERM domain may render the RTX mutants with the nonfunctional structures less able to interact with the host cell.
中文摘要 I-II
Abstract III-IV
致謝 V
表目錄 VIII
圖目錄 IX
符號與縮寫 X
緒論 1-7
I. 實驗菌株與質體 8
II. 實驗菌種的培養與保存方法 8
III. 實驗方法 8
1. 商業化套組純化質體DNA 8
2. 聚合脢連鎖反應 9
3. DNA 電泳分析 9
4. 限制酶切割DNA 9
5. DNA片段之分離與回收 10
6. DNA片段之去磷酸化反應 10
熱休克轉形作用 11
接合生殖作用 11
蔗糖培養基與抗生素培養基之篩選 12
細菌生長曲線之測定 13
細菌全細胞可溶性蛋白質之萃取 13
蛋白質樣品之定量 13
硫酸十二酯鈉聚丙烯醯胺膠體電泳 14
蛋白質染色 14
西方墨點法(Western-blotting assay) 14
實驗細胞株的培養與保存方法 15
細胞毒性實驗 (Cytotoxicity test) 15
吖啶橙-結晶紫染色 (Acridine orange-crystal violet stain) 16
肌動蛋白交聯試驗 (Actin cross-linking assay) 17
免疫螢光染色 17
結果 19-22
討論 23-27
參考文獻 28-32

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