| 摘要: | 創傷弧菌(Vibrio vulnificus)是一株生活在海水的高致死率致病菌，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。由於創傷弧菌好發於肝臟相 關疾病及免疫力低弱的患者，且台灣肝病患者的比率相當高，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人類的死亡，不外乎是致病菌產生毒素而造成宿主細胞的死亡，進而傷害器官的正常功能而出現嚴重的疾病症狀，造成人類的死亡。已知在致病菌感染人類時，他們往往可以逃過宿主免疫系統的破壞，造成感染人類的死亡。巨噬細胞是人體體內的非專一性免疫細胞，扮演著人體第一道的防線，抵禦致病菌對人體的傷害。近來的研究指出，創傷弧菌在感染小鼠初期，會藉由破壞小鼠體內的巨噬細胞來達到感染的目的。因此，我們試圖找出創傷弧菌毒殺巨噬細胞的毒殺因子，以進一步瞭解創傷弧菌毒殺巨噬細胞的毒殺機制，以解決創傷弧菌的感染問題。 日前，我們已由轉位子突變株庫中分離並確認一個新穎的巨噬細胞毒殺基因-細胞毒素基因(hly)。在過去的研究計畫結果顯示，創傷弧菌hly 對巨噬細胞及小鼠具有毒殺的能力。在螢光顯微鏡顯示，以GFP標誌的Hly (Hly-GFP)會作用到巨噬細胞的細胞膜。以 PEG 的保護分析顯示，創傷弧菌Hly可能是透過對巨噬細胞形成穿孔的現象，而引起細胞的死亡。另外，由propidium iodide 及annexin V染色及pan-caspase inhibitor zVAD 的分析，顯示創傷弧菌hly 主要是引起巨噬細胞細胞壞死的死亡型態，並不是細胞的凋亡。 在本研究計畫中，為了進一步釐清創傷弧菌細胞毒素基因(hly)在致病機轉的角色，我們試圖進行分析(1)創傷弧菌細胞毒素(hly)以Ca2+訊號引發巨噬細胞死亡的訊息傳遞途徑，(2)創傷弧菌細胞毒素以 p53訊號引發巨噬細胞死亡的訊息傳遞途徑，以了解細胞毒素基因(hly)在感染過程的角色，分析細胞毒素基因(hly)作為治療創傷弧菌感染藥物設計的標的蛋白，能更有效更快速地抑制細胞急速死亡，以避免組織壞死及過度發炎所引起的死亡，以達到治癒效果，並進一步確認細胞毒素(Hly)做為疫苗的可行性，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。 目前完成的研究結果顯示，創傷弧菌細胞毒素基因hly會引發巨噬細胞外的Ca離子流入胞內的粒線體，進而影響NADPH oxidase及蛋白酶calpain，產生大量的ROS並破壞粒線體，導致巨噬細胞的程序式死亡作用。
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. In order to solve the problem of the pathogen infection, it is essential to understand its pathogenesis. Several bacterial products such as capsule polysaccharide (CPS), iron-acquestering systems, type IV leader peptidase-N-methyltransferase, K+ uptake protein TrkA, and RTX toxin have been suggested as potential virulence factors of Vibrio vulnificus; however, the pathogenic mechanism of V. vulnifiucs infection has not been fully delineated. In human body, macrophage is an important defense phagocyte 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, 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. In previous project, we report that one of the isolated Tn mutants defective in the macrophage cytotoxicity had an insertion within an open reading frame identified as hly, a gene encoding a putative pore protein from V. vulnificus. The cytotoxin gene (hly) mutant of V. vulnificus was used to characterize the mechanism for cell death induced by hly of V. vulnificus in macrophages. The hly gene exhibits a significant cytotoxicity to macrophages. The cytopathic effect is characterized as necrosis rather than apoptosis based on annexin V and propidium iodide staining and pan-caspase inhibitor zVAD treatment. The Hly toxin caused cell lysis through formation of membrane pores. Taken together, these results indicate that V. vulnificus cytotoxin (Hly) toxin elicits the necrotic death through formation of membrane pores in macrophage. In this project, we attempt to 1. identify the pathway of macrophage death elicited by hly in V. vulnificus through Ca2+ signal 2. identify the pathway of macrophage death elicited by hly in V. vulnificus through p53 In this project, we report that V. vulnificus cytotoxin gene (hly) triggers a programmed cell death in macrophages through Ca2+/ROS-mediated mitochondrial dysfunction. |
