溶血素及非溶血素基因之溶血活性鑑定及生化特性研究

Abstract

溶血素在弧菌致病的過程中被認為是很重要的毒性因子，在致病的過程中可以藉由溶血來破壞細胞膜而達到入侵的效果。除了溶血素蛋白具有溶血的功能外，一些毒性蛋白也擁有溶血的活性。寡胜肽膜透酶 (oligopeptide permease A) 為一種可與ATP結合並從事運輸的膜蛋白。本研究在研究運輸蛋白的特性過程中發現，此運輸蛋白除了具有運輸的功能外，還具有溶血素的活性。此現象在運輸蛋白及溶血素的文獻中為首次被發現。我們利用純化的方法及生化特性分析，證實此蛋白在不同動物的紅血球中，有特定的偏好性。並對中國蒼鼠卵巢癌細胞K1 (CHO-K1) 具有毒殺性及可改變細胞的形態。我們利用質譜儀分析所純化的蛋白質N端及內部的序列，並利用分子生物的技術鑑定出此蛋白序列為運輸蛋白的基因，並將完整基因選殖出來。並且利用同源重組法構建弗尼斯氏弧菌hly-oppA基因缺失突變株。探討並分析一系列的生化特性影響，包括：寡胜肽的結合能力、螢光受質的運輸效率、抗生素的敏感性測式、生物膜的生成及細菌形態上的改變。此外與野生株比較，對BALB/c小鼠而言，hly-oppA基因缺失突變株不具有致死傷害性。另外，我們也利用分子選殖技術從其它弧菌中，將其相似性的基因選殖並表現分析活性。發現此一類蛋白具有多重功能的特性，在弧菌中並非特定現象。 除此之外，為了探討及比較溶血素及非溶血素基因所引起之溶血活性的生化功能特性，我們選擇熱穩定性溶血素做為研究目標。我們從海洋弧菌中首次發現熱穩定性溶血素之基因，並對其進行蛋白質表現，純化分離及其特性之探討。在生物特性分析上，藉由所純化分離的溶血素重組蛋白分析，證實此蛋白在不同動物的紅血球中，有特定的偏好性。並對動物細胞具有毒殺性及可改變細胞的形態。對BALB/c小鼠具有致死性的組織性傷害。我們利用飽合定點突變的技術及蛋白質結合分子探針的方法，探討並分析熱穩定性溶血素在毒性功能方面的作用。在物理特性分析上，藉由所純化分離蛋白質分析，證實此蛋白具有Arrhenius的現象，此現象為將蛋白質加熱至60-75 ℃的溫度時，蛋白質會失去毒性的作用;但將其繼續加熱至80 ℃以上的溫度並迅速冷卻至4 ℃的溫度時，此蛋白質會恢復其毒性功能。我們利用超高速離心及穿透式電子顯微鏡分析此一現象，發現此蛋白質存在著二種構造形態，其中一種是自然形態的構形具有毒性作用;另一種則是當加熱後會形成纖維狀的構形不具有毒性作用。我們也利用差異性掃描熱量儀分析蛋白質的結構穩定性。另外，我們也利用懸吊液滴蒸汽擴散法進行蛋白質晶體的培養，並收集完整X光機的數據及決定蛋白質晶體的空間群屬及晶格的參數。除此之外，我們也發現此熱穩定性溶血素具有去氧核醣核酸水解酶的第二種功能，可以對DNA進行水解並且可以影響細胞走向自殺途徑。

Hemolysins have been suggested to be an important virulence factor in the pathogenic processes of many vibrio species. They invade the host by hemolysis to disturbing the membranes in the pathogenic process. In addition to hemolysins have possessed of hemolysis, some toxins protein also have exhibiting hemolytic activity. In our study, we identified and characterized of an ATP-binding cassette type transporter protein, oligopeptide permease A, with hemolytic activity, are detailed, representing the first example that a transporter protein can possess both the transporter function and hemolysin activity. Biological properties, associated with hemolytic activities, were demonstrated with the purified and recombinant proteins, which exhibited host-specific hemolytic activity, on various mammalian erythrocytes, and apparent cytotoxicity and morphological change on the CHO-K1 cells. The transporter identity was revealed by both the N-terminal and internal peptide sequences of the purified protein and confirmed by the molecular cloning of the corresponding gene and functional expression of the recombinant protein, as well as construction of the V. furnissii hly-oppA knockout mutant, which showed apparent effects on oligopeptide binding, fluorescent substrate transport efficiency, antibiotic susceptibility, biofilm production amount, and cell morphology change. The mortality rate determination of the V. furnissii wild-type and the hly-oppA knockout mutants on the BALB/c mice indicated the effect of the Hly-OppA, in the virulence of V. furnissii. Finally, the ubiquitous determination of hemolytic activity from the various homologous ABC type transporter proteins suggested the biological role of the Hly-OppA protein, in virulence. Additionally, we also identified the TDH protein and characterized its biochemical activity for hemolytic activity emerges from hemolysin and non-hemolysin gene. We have identified a thermostable direct hemolysin (tdh) gene from Vibrio vulnificus. We have expressed, purified and characterized the corresponding TDH protein. Biological properties associated with hemolytic activities were demonstrated with the purified recombinant protein, which exhibited host-specific hemolytic activity on various mammalian erythrocytes. In addition, apparent cytotoxicity and morphological change on mammalian cells, contributes to mortality and tissue damage, in BALB/c mice. Site-saturated mutagenesis techniques and molecular probes were also used to analyze functions in the toxic effect of TDH. Biophysical properties associated with the Arrhenius effect were demonstrated with the purified protein, which exhibited de-toxicity by heating at 60-75 ℃ but would be reactivated by additional heating above 80 ℃. Analytical ultracentrifugation and transmission electron microscopy (TEM) image analysis revealed that TDH possesses two protein forms: one is native and the other is the fibril-form. Differential scanning calorimetry (DSC) was also used to investigate protein stability. Moreover, the TDH protein was subjected to structural determination and crystallized via crystallization using the hanging-drop vapor-diffusion method, X-ray data collection, and space group determination. Finally, the discovery of nuclease activity might potentially indicate a second functional role of TDH protein in cell apoptosis.