致病性坏死梭杆菌溶胶原蛋白提取与毒性分析

Purification and toxicity analysis of a collagenolytic protein from the highly pathogenic Fusobacterium necrophorum

为验证坏死梭杆菌的溶胶原活性以及溶胶原蛋白在细菌感染宿主早期的可能作用,本研究利用超声波破碎处理、离子交换层析和凝胶过滤技术,从坏死梭杆菌国内流行株(AB株)的菌体分离获得一种具有溶解牛Ⅱ型胶原蛋白能力的溶胶原活性蛋白(CP)。CP的相对分子量约为50 ku,CP的胶原酶活力为20.3 U/mL。将其腹腔接种小鼠,纯化的CP可诱导小鼠出现皮下坏死病灶,肝、脾脏肿大等症状;组织病理学分析表明CP导致小鼠肝细胞出现大面积坏死,并形成肉芽肿病症;另外,CP对小鼠肝细胞(NCTC 1469)表现出细胞毒性作用,出现多种超微结构变化特征,但小鼠巨噬细胞(Ana-1)对CP的敏感性相对较弱。研究结果表明,本研究分离获得的CP的生物学特性与已报道的坏死梭杆菌毒力因子的生物学特性截然不同,推测CP可能是一种新型毒力因子,可能在细菌感染宿主早期入侵宿主过程中发挥重要作用。

To study the pathogenic mechanism of Fusobacterium necrophorum in early stage of infection, a collagenolytic protein （approximately 50 ku） was purified from the ultrasonic extract of F.necrophorum. The purified coUagenolytic protein caused death or various signs of illness in mice. Necropsy of inoculated mice revealed hepatomegaly, splenomegaly and necrotic lesions at the site of injection. Histopathological analysis of the liver of infected mice showed evidence of coagulative necrosis and microabscess formation. Furthermore, It also induced a variety of signs of cytopathic effect （CPE） on liver cell of mouse （NCTC 1469）, including loss of surface projection, overall smoothening, decreased cell size and organelle condensation. However, purified collagenolytic protein showed less cytotoxicity to peritoneal macrophage of mouse （Ana-1）, not like any other known bacterial virulence factors of F.necrophorum. So the collagenolytic protein was postulated to be a novel virulence factor and may play important role in the pathogenic mechanism of F.necrophrum.