| 摘要: | 創傷弧菌（Vibrio vulnificus）是一株生活在海水中的高致死率致病菌，且其發病到死亡往往只要2-3 天，在臨床的病徵主要是食入含有創傷弧菌的海產所引發的敗血症，或是經由外傷感染，包括海產養殖器具或海產刺傷所造成的組織潰爛。因為創傷弧菌好發於肝臟相關疾病以及免疫力低弱的患者，並且台灣肝病患者比率相當高，再加上台灣養殖業興盛且近年來感染病例持續增加，因此，尋找創傷弧菌主要的致病基因，以進一步加以控制並治療此致病菌所造成的傷害更是刻不容緩。目前致病菌之所以會引起寄主細胞的死亡，不外乎是致病菌感染寄主細胞或是致病菌產生毒素而造成細胞的死亡，寄主細胞的死亡往往會傷害正常的器官功能而出現疾病的症狀，已知健康的細胞受到刺激而引起細胞死亡的型態有數種，包括apoptosis, pyroptosis, oncosis 及autophagy。致病菌在感染人類時，往往可以逃過寄主免疫系統的破壞，然而macrophage 是非專一性免疫細胞，常扮演著第一道防線，近年來有許多研究報告指出，許多致病菌（如Shigella、Salmonella 及Yersinia）能藉由細胞毒素而導致 macrophage 進行凋亡（apoptosis），以達到感染的目的。有關V. vulnificus 與phagocytes 的關係大多侷限在capsule，具有capsule 的V. vulnificus 比capsule 缺失的菌株對 macrophage 的吞噬作用較有抗性，近來的研究指出，不論是在細胞株macrophage J774 或是在受到V. vulnificus 感染的小鼠的腹腔macrophage，V. vulnificus 均會導致macrophage 進行細胞的凋亡現象，除此之外，也有報告指出V. vulnificus 會在感染小鼠初期，會藉由破壞macrophage 來達到感染的目的，然而，目前有關V. vulnificus 毒殺macrorphage 的機制仍然尚未清楚。由上所述，我們認為創傷弧菌之所以可以感染人類，主要是創傷弧菌會毒殺巨噬細胞，以達到感染的目的。因此，我們試圖找出創傷弧菌毒殺巨噬細胞的相關基因，以進一步瞭解致病毒殺巨噬細胞的毒殺機制，以解決創傷弧菌的感染問題。在本研究計畫中，為了要尋找出創傷弧菌對巨噬細胞的毒殺基因，我們試圖 (1) 建構轉位子突變株庫並藉由細胞毒殺分析由轉位子突變株庫中篩選對macrophage 毒殺能力下降的轉位子突變株 (2) 由macrophage 毒殺能力下降的轉位子突變株選殖對macrophage 的毒殺基因 (3) 分析macrophage 毒殺基因毒殺細胞的毒殺途徑目前，我們已完成構築轉位子突變株庫，共有6,548 株突變株，並由2,481 株轉位子突變株進行對macrophage RAW264.7 的毒殺能力實驗分析，找出9 株對macrophage RAW264.7 的毒殺能力下降的轉位子突變株，經由這些對macrophage RAW264.7 的毒殺能力下降的突變株中，選殖並分析這些細胞毒殺基因可能的功能，將這9 株突變株經由基因的選殖及序列的分析，顯示這些巨噬細胞毒殺基因的功能包括：GTPase 活性（ychF）、第二型蛋白分泌能力（epsD 及epsM）、細胞毒殺能力（rtxA、vvhA 及vvhB）、以及一個功能未知的基因（rtxH）。其中，創傷弧菌對巨噬細胞的毒殺因子ychF 與大腸桿菌（E. coli K-12）的GTP 結合蛋白YchF 具有79%相同性，與耶爾森氏菌（Yersinia pseudotuberculosis IP32953）的老鼠致病因子VagA 具有78%相同性，而與布魯氏菌（Brucella melitensis 16M）在調控鐵利用的DugA 具有56%相同性。經本實驗室的研究結果顯示，創傷弧菌YchF 除了扮演對巨噬細胞毒殺的角色外，也同時具有對鐵利用的功能。因此，在本研究計畫中，我們要繼續(I)由這些轉位子突變株，藉由釋出的LDH 活性分析來篩選對macrophage RAW264.7 的毒殺能力下降的突變株，(II)並進一步選殖對 macrophage RAW264.7 的毒殺基因，(III)釐清這些細胞毒殺基因的毒殺途徑（機制），以了解macrophage 毒殺基因在感染的角色，期望能尋找出有效的致病因子當作設計殺菌藥物的標的蛋白，能更有效更快速殺死創傷弧菌或抑制其快速生長以達到治癒效果，並進一步尋找有效抗原蛋白以製備疫苗，以期能達到預防創傷弧菌感染的目的，並提供研究人員對其他致病菌在細胞毒殺的機制有所瞭解，可以在致病菌的感染控制上有所助益。
Vibrio vulnificus, a highly virulent marine bacterium, is the causative agent of seafood-related diseases, such as primary septicemia and wound infection in human, particularly in those with certain underlying diseases especially in patients with chronic liver disease. Cases of V. vulnificus infections have been reported from many areas of the world. Over the last decade, there has been a dramatic increase in the number of cases due to V. vulnificus in the southern part of Taiwan. Because V. vulnificus often contaminates the seafood and the consumption of seafood especially raw seafood is very popular, V. vulnificus infection is apparently more prevalent on this island than in many areas of the world. Several bacterial products such as capsule polysaccharide (CPS), iron-acquestering systems, type IV leader peptidase-N-methyltransferase, K+ uptake protein TrkA, and RTX toxin (20, 24) have been suggested as potential virulence factors of Vibrio vulnificus; however, the pathogenic mechanism of V. vulnifiucs infection has not been fully delineated. In addition, a correlation between the presence of two exotoxins, hemolysin/cytolysin and metalloprotease, and the virulence of V. vulnificus has been reported, although these two exotoxins were not confirmed as virulence factors by genetic analysis. In order to solve the problem of the pathogen infection, it is essential to understand its pathogenesis. Macrophage is an important defense mechanism in the host against invading microorganisms. In response to this host defense, V. vulnificus, like many pathogenic bacteria, may evolve strategies to counter the bactericidal effect of macrophage. Recently, Kashimoto et al. indicated that clinical isolates of V. vulnificus exhibit apoptotic activity toward macrophage, but environmental isolates could not induce apoptosis in vitro and in vivo. In addition, Tsuchiya et al. also indicated that V. vulnificus infection progresses by damage to macrophages during the early phase of infection. Taken together, we suggested that the mechanism by which V. vulnificus kills macrophage is important for causing serious infection. However, a comprehensive analysis of gene(s) contributing cytotoxicity toward macrophage has not been conducted. In this study, in order to identify and characterize the gene(s) required for cytotoxicity toward macrophage, we attempt to (1) construct a transposon (Tn) insertional mutant library and screen them by a macrophage cytotoxicity assay (2) isolate Tn mutants defective in macrophage cytotoxicity and clone these genes required for cytotoxicity to macrophage from these Tn mutants (3) identify the death pathway of macrophage elicited by these cytotoxicity genes In the present, we already constructed a Tn mutant library (6,548 mutants) and screened 2,481 Tn mutants by cytotoxicity assay, and isolated 9 Tn mutants exhibited decreased cytotoxic activity toward marcrophage compared to wild type. The nine genes inserted by transposon were cloned and sequenced from 9 Tn mutants. Homology comparison of protein sequence revealed three kinds of putative function: GTPase activity, protein secretion, and cytotoxic activity. We report that one of the isolated Tn mutants defective in the macrophage cytotoxicity had an insertion within an open reading frame identified as ychF, a gene encoding a putative GTPase protein from V. vulnificus. V. vulnificus YchF was 58% of sequence identity to that of DugA of Brucella melitensis 16M, a protein required for the acquisition of iron. At Fe3+-limiting LB broth, the ychF mutant of V. vulnificus showed attenuated growth compared to the wild-type strain. The ychF mutation resulted in deficient in cytotoxicity to macrophage and acquisition of iron, which were fully restored by in trans complementation. Additionally, ychF was not required for intracellular survival within macrophage but also could not cause cytotoxic to HeLa cells in V. vulnificus. These results demonstrate that V. vulnificus ychF was associated with cytotoxicity toward macrophage and acquisition of iron but not required for intracellular survival within macrophage. |
