Obiso R J, Lyerly D M, Van Tassell R L, Wilkins T D
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg 24061-0305, USA.
Infect Immun. 1995 Oct;63(10):3820-6. doi: 10.1128/iai.63.10.3820-3826.1995.
Strains of Bacteroides fragilis that produce an enterotoxin have been implicated in diarrheal disease in farm animals and humans during the past decade. Our laboratory has purified and characterized this enterotoxin as a single polypeptide (M(r), approximately 20,000). Recently, we used PCR to clone and sequence the enterotoxin gene from B. fragilis and showed that it exhibits significant homology with extracellular metalloproteases. Further studies showed that the purified enterotoxin has protease activity. To further characterize the role of this enterotoxin in diarrheal disease, we studied the histological and pathological effects of highly purified B. fragilis enterotoxin in lamb, rabbit, and rat ligated intestinal loops. When the enterotoxin was injected into ligated ileal and colonic loops, there was significant tissue damage and subsequent fluid accumulation. The fluid response in the ileum was greater in lambs than in rabbits and rats, whereas the fluid response in the colon was greater in rabbits than in lambs and rats. Analysis of the intestinal fluid elicited by the enterotoxin revealed an accumulation of chloride and sodium as well as albumin and total protein. Histological examination revealed mild necrosis of epithelial cells, crypt elongation, villus attenuation, and hyperplasia. There was extensive detachment and rounding of surface epithelial cells and an infiltration of neutrophils. Enterotoxic activity was inhibited by the metal chelators EDTA and 1,10-phenanthroline; to some degree, the enterotoxic activity could be reconstituted by the addition of zinc to the chelated enterotoxin. Our results indicate that the enterotoxin elicits a significant fluid response subsequent to tissue damage in the small and large intestine. These data further support the idea that this enterotoxin is an important virulence factor in B. fragilis-associated diarrhea.
在过去十年中，已发现产生肠毒素的脆弱拟杆菌菌株与农场动物及人类的腹泻病有关。我们实验室已将这种肠毒素纯化并鉴定为一种单一多肽（相对分子质量约为20,000）。最近，我们利用聚合酶链反应（PCR）从脆弱拟杆菌中克隆并测序了肠毒素基因，结果表明它与细胞外金属蛋白酶具有显著同源性。进一步研究表明，纯化后的肠毒素具有蛋白酶活性。为了进一步阐明这种肠毒素在腹泻病中的作用，我们研究了高纯度脆弱拟杆菌肠毒素对羔羊、兔子和大鼠结扎肠袢的组织学和病理学影响。将肠毒素注入结扎的回肠和结肠肠袢后，出现了明显的组织损伤及随后的液体蓄积。羔羊回肠的液体反应比兔子和大鼠更强烈，而兔子结肠的液体反应比羔羊和大鼠更强烈。对肠毒素引发的肠液分析显示，其中氯化物、钠、白蛋白和总蛋白均有蓄积。组织学检查发现上皮细胞轻度坏死、隐窝延长、绒毛变细以及增生。表面上皮细胞广泛脱落并呈圆形，且有中性粒细胞浸润。金属螯合剂乙二胺四乙酸（EDTA）和1,10 - 菲咯啉可抑制肠毒素活性；在一定程度上，向螯合后的肠毒素中添加锌可恢复其肠毒素活性。我们的结果表明，肠毒素在小肠和大肠组织损伤后引发了显著的液体反应。这些数据进一步支持了这种肠毒素是脆弱拟杆菌相关腹泻中一种重要毒力因子的观点。
