TRICHOMONAD SURFACE AND EXTRACELLULAR PROTEINASE

毛滴虫表面和细胞外蛋白酶

• 批准号: 3147712
• 负责人: LYNETTE Bundy CORBEIL
• 金额: $ 14.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-06-01 至 1995-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3147712
• 关键词: SDS polyacrylamide gel electrophoresis; Trichomonas; Trichomonas vaginalis; antibody formation; antigen antibody reaction; cow; endopeptidases; enzyme activity; enzyme linked immunosorbent assay; gene expression; host organism interaction; image processing; immunoglobulin G; ion exchange chromatography; laboratory mouse; laboratory rabbit; molecular cloning; neutralizing antibody; nucleic acid probes; pathologic process; protein structure function; protozoal antigen; protozoal infection; radionuclides; reproductive system disorder; restriction mapping; southern blotting; virulence; western blottings

The long term goal of this research is to elucidate the pathogenic mechanisms of and protective host responses to trichomonad membrane and extracellular proteinases. Trichomoniasis is a prevalent sexually transmitted human and bovine disease for which there is no laboratory animal model of reproductive tract infection. Tritrichomonas foetus proteinase was chosen for study because of evidence that it is a virulence factor. Moreover, this organism offers a unique opportunity to test potential immune defense mechanisms in vivo in the natural host, cattle. Recently we showed that the T. foetus extracellular proteinase cleaves proteins involved in defense of the reproductive tract, such as IgG1, IgG2, lactoferrin, fibronectin and fibrinogen. After characterizing the extracellular proteins (which is also present in T. foetus membranes), we have partially purified the enzyme. Therefore, we are now in a position to further characterize its role in virulence and the ability of antiproteinase antibody to neutralize enzymatic activity. The latter is of special interest because others have shown antiproteinase antibody to protect sheep against disease caused by Bacteroides nodosus. To further study the role of the T. foetus extracellular/membrane proteinase in pathogenesis and the role of antibodies to proteinase protection, we propose to: 1) Investigate specific potential pathogenic mechanisms of trichomonad proteinase (ability to cleave different allotypes of IgG2, to lyse nucleate cells or rbcs and to promote adherence of T. foetus to cells). 2) Clone, express and characterize the gene(s) for T. foetus proteinase. This will include Southern blots to determine whether the gene is conserved in T. foetus isolates and whether it is present in T. vaginalis. 3) Produce and characterize antibody against T. foetus semipurified and/or recombinant proteinase. 4) Determine whether antibodies to T. foetus proteinase will neutralize the enzyme and interfere with functions correlated with virulence. If appropriate, ability to neutralize T. Vaginalis proteinase will be tested also. This research should provide new insights into virulence mechanisms of T. foetus and host defense mechanisms. Since T. vaginalis also produces extracellular proteinase, the data may have broader application to an important human sexually transmitted infection.

这项研究的长期目标是阐明致病因素 毛滴虫膜的机制和保护性宿主反应 细胞外蛋白酶。滴虫病是一种常见的性病 没有实验室可检测的人类和牛传播疾病 生殖道感染动物模型。胎儿三滴虫 选择蛋白酶进行研究是因为有证据表明它是一种 毒力因子。此外，这种有机体提供了一个独特的机会 测试自然宿主体内潜在的免疫防御机制， 牛。最近我们发现胎儿毛蝽细胞外蛋白酶 裂解参与生殖道防御的蛋白质，例如 IgG1、IgG2、乳铁蛋白、纤连蛋白和纤维蛋白原。后 表征细胞外蛋白（也存在于 T. 胎儿膜），我们已经部分纯化了酶。因此，我们 现在能够进一步描述其在毒力和 抗蛋白酶抗体中和酶活性的能力。 后者特别令人感兴趣，因为其他人已经表明 抗蛋白酶抗体可保护羊免受由以下疾病引起的疾病 结节拟杆菌。进一步研究 T. fetus 的作用 细胞外/膜蛋白酶在发病机制中的作用及其作用 针对蛋白酶保护的抗体，我们建议： 1) 研究滴虫的具体潜在致病机制 蛋白酶（能够裂解 IgG2 的不同同种型，裂解 有核细胞或红细胞并促进胎儿毛滴虫与细胞的粘附）。 2) 克隆、表达并表征胎儿毛蚴蛋白酶的基因。 这将包括 Southern 印迹以确定该基因是否是 在 T. fetus 分离株中保守，以及它是否存在于 T. fetus 中。 阴道炎。 3) 产生并表征针对 T. fetus 半纯化的抗体 和/或重组蛋白酶。 4) 确定胎儿毛毛虫蛋白酶的抗体是否会中和 酶并干扰与毒力相关的功能。如果 适当的，中和阴道毛滴虫蛋白酶的能力将是 也测试过。 这项研究应该为病毒的毒力机制提供新的见解。 T.胎儿和宿主的防御机制。由于阴道毛滴虫还产生 细胞外蛋白酶，该数据可能具有更广泛的应用 人类重要的性传播感染。