PHYSIOLOGY AND CLONING OF B FRAGILIS ENTEROTOXIN

脆弱拟杆菌肠毒素的生理学和克隆

• 批准号: 3246992
• 负责人: CYNTHIA SEARS
• 金额: $ 19.21万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-20 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3246992
• 关键词: Bacteroides; actins; bacterial toxicology; chlorine; electron microscopy; enterotoxins; gastrointestinal infection; immunofluorescence technique; ion transport; keratin; light microscopy; membrane potentials; molecular cloning; nucleic acid probes; nucleic acid sequence; physiology; tissue /cell culture; tubulin; virulence

Enterotoxigenic Bacteroides fragilis (ETBF) have been isolated from the feces of 10 to 20% of diarrheic livestock and is a newly recognized human enteric pathogen. In both naturally and experimentally infected animals, the predominant site of altered histopathology has been the colon. To date, conventional tissue culture and animal assays used to detect the biologic activity of bacterial enterotoxins have failed with ETBF. We have developed a tissue culture assay using the cloned human colonic epithelial cell line, HT29/C1, which is 89% sensitive and 100% specific in detecting ETBF strains as defined by the lamb ligated intestinal loop assay. Subconfluent HT29/C1, cells treated with less than 1 nanogram of the purified ETBF heat-labile protein toxin rapidly develop dramatic morphologic changes with rounding and detachment from adjacent cells. These in vitro morphologic changes mimic those observed in vivo in natural and experimental infection. Furthermore, in polarized confluent cell monolayers, the transepithelial resistance of the monolayers is diminished and electrogenic active chloride secretion is stimulated without any alteration in cell viability. These data indicate that the ETBF toxin is an enterotoxin and a nonlethal cytotoxin. We hypothesize that the ETBF toxin is a key virulence factor of B. fragilis strains associated with diarrheal disease and that this toxin alters the intestinal epithelial barrier by effecting a change in cell shape resulting in disruption of tight junctions. Intestinal secretion may result from leakiness of the paracellular transport pathway and direct stimulation of transcellular chloride secretion. To address this hypothesis, the specific aims of this project are: I. To identify the cytoskeletal effects of the ETBF toxin. The morphologic changes induced by ETBF and its toxin in HT29/C1 cells and lamb intestine over time will be characterized by light and electron microscopy; nd by studies of the cytoskeletal proteins, actin, tubulin and keratin. II. To investigate the effects of the ETBF toxin on cellular function. The time course and mechanism(s) by which the ETBF toxin diminishes the transepithelial resistance and stimulates chloride secretion in polarized monolayers of HT29/C1 cells will be examined; and III. To establish the importance of the ETBF toxin as a virulence factor. The ETBF toxin gene will be cloned and the importance of this toxin in the pathogenesis of ETBF infections will be established by studies utilizing constructed isogenic strains and by use of an ETBF toxin DNA probe in human epidemiologic investigations. These studies utilizing a human intestinal epithelial cell line and lamb intestine will begin to characterize by cell biology, physiology and genetic techniques the importance in disease pathogenesis of the ETBF toxin, a newly identified virulence factor of B. fragilis.

产肠球菌脆弱拟杆菌（ETBF）已从 粪便的10至20%的蚯蚓牲畜，是一种新认识的人类 肠道病原体 在自然感染和实验感染的动物中， 组织病理学改变的主要部位是结肠。 到 日期，常规组织培养和动物试验用于检测 细菌肠毒素的生物学活性已经用ETBF失效。 我们 已经开发了一种组织培养试验， 上皮细胞系HT 29/C1，敏感性89%，特异性100% 在检测由羔羊结扎肠袢定义的ETBF菌株中， 比色法 亚融合的HT29/C1，用少于1纳克的H2O2处理的细胞 纯化ETBF热不稳定蛋白毒素迅速显着发展 形态学改变，变圆并与相邻细胞分离。 这些体外形态学变化模拟了在体内观察到的那些， 自然和实验感染。 此外，在极化融合中， 细胞单层，单层的跨上皮电阻是 减少和刺激产电活性氯化物分泌 而细胞活力没有任何改变。 这些数据表明 ETBF毒素是一种肠毒素和非致死性细胞毒素。 我们假设 ETBF毒素是B的关键毒力因子。fragilis菌株 这种毒素会改变 肠上皮屏障通过影响细胞形状的变化 导致紧密连接的破坏。 肠道分泌物可能 由细胞旁转运途径的泄漏和直接 刺激跨细胞氯分泌。 为了解决这个 假设，本项目的具体目标是：一。识别 ETBF毒素的细胞骨架效应。 诱导的形态学变化 随着时间的推移，ETBF及其毒素在HT29/C1细胞和羔羊肠中的作用将 通过光学和电子显微镜表征;并通过研究 细胞骨架蛋白、肌动蛋白、微管蛋白和角蛋白。 二.探讨 ETBF毒素对细胞功能的影响 时间进程和 ETBF毒素减少经上皮细胞 电阻和刺激氯分泌极化单层 将检查HT29/C1细胞;和III.确立…的重要性 ETBF毒素作为毒力因子。 ETBF毒素基因将被克隆 以及这种毒素在ETBF感染发病机制中的重要性 将通过利用构建的等基因菌株的研究来建立， 在人类流行病学调查中使用ETBF毒素DNA探针。 这些研究利用人肠上皮细胞系和羔羊 肠将开始以细胞生物学、生理学 遗传技术在ETBF发病机制中的重要性 毒素，一种新发现的B的毒力因子。脆弱的