CHARACTERIZATION OF THE TOXINS OF BACTERIOIDES FRAGILES

脆性拟杆菌毒素的表征

• 批准号: 3148024
• 负责人: Tracy D. Wilkins
• 金额: $ 21.39万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-01 至 1995-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3148024
• 关键词: Bacteroides; antigen receptors; bacterial disease; bioassay; carbohydrate receptor; diarrhea; enterotoxins; enzyme linked immunosorbent assay; gastrointestinal disorder diagnosis; goats; human tissue; laboratory rabbit; molecular cloning; monoclonal antibody; nucleic acid probes; nucleic acid sequence; protein purification; sheep; technology /technique development

Bacteroides fragilis is a species of anaerobic bacteria which lives in the colon of humans and animals. It makes up only about 1% of the human colonic flora, however, it is the most commonly isolated anaerobe from abscesses and soft tissue infections. Recent reports have shown that certain strains of B. fragilis produce an enterotoxin, suggesting a new role for this organism as an enteropathogen as well as evidence for a new virulence factor involved in its pathogenicity in tissues. This toxin has been implicated as a cause of diarrhea in several species of farm animals and more recently in humans, particularly infants. In preliminary studies, it appears that about 10% of isolates produce the toxin, suggesting that it is present in the colon in many individuals over long periods of time. The activity of the toxin was first discovered in the mid-1980's using the ligated ileal loop assay in lambs. Then, in 1990, it was shown that the activity could be detected by tissue culture assay using HT-29 colon carcinoma cells. We have now purified the toxin to homogeneity and showed that the enterotoxic activity observed in the lamb ligated loop assay and the cytotoxic activity (i.e., rounding of HT-29 cells) is due to the same protein. We have characterized the toxin, which has an M of about 20,000 and determined a partial N-terminal amino acid sequence. We are now cloning and sequencing the toxin gene, and studying its receptors and mechanism of action. We are also making specific polyclonal and monoclonal antibodies against the toxin to develop specific ELISA assays for detecting the toxin in feces and other clinical samples. This will establish a firm base for future collaborations with clinical investigators to determine the role of this toxin in etiology of diarrheal and infectious diseases and to determine approaches to diagnosis, therapy and treatment.

脆弱拟杆菌（Bacteroides fragilis）是一种厌氧细菌，生活在 人类和动物的结肠。 它只占人类的1%左右 然而，它是结肠菌群中最常见的分离厌氧菌 脓肿和软组织感染。 最近的报告表明 脆弱拟杆菌的某些菌株会产生肠毒素，这表明存在一种新的 该生物体作为肠道病原体的作用以及新的证据 毒力因子参与其在组织中的致病性。 这种毒素有 被认为是多种农场动物腹泻的原因 最近在人类，尤其是婴儿中也出现了这种情况。 在初步研究中， 似乎大约 10% 的分离株产生这种毒素，表明它 长期存在于许多人的结肠中。 这 该毒素的活性首次在 20 世纪 80 年代中期被发现 羔羊结扎回肠环测定。 然后，在 1990 年，研究表明 可以使用 HT-29 结肠通过组织培养测定来检测活性 癌细胞。 我们现在已将毒素纯化至同质并显示 在羔羊结扎环试验中观察到的肠毒性活性 细胞毒活性（即 HT-29 细胞的四舍五入）是由于相同的 蛋白质。 我们已经对毒素进行了表征，其 M 约为 20,000 并确定了部分N-末端氨基酸序列。 我们现在 克隆和测序毒素基因，并研究其受体和 作用机制。 我们还正在制作特定的多克隆和单克隆 针对毒素的抗体以开发特异性 ELISA 检测方法 粪便和其他临床样本中的毒素。 这将建立一个坚定的 为未来与临床研究人员合作确定基础 该毒素在腹泻和传染病病因学中的作用以及 确定诊断、治疗和治疗的方法。