CAMPYLOBACTER JEJUNI CYTOTOXINS

空肠弯曲杆菌细胞毒素

基本信息

批准号：
3455144
负责人：
CAROL L PICKETT
金额：
$ 10.14万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
1990
资助国家：
美国
起止时间：
1990-01-01 至 1994-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3455144
关键词：
Escherichia coli bacterial genetics bacterial toxins chickens disease /disorder model genetic manipulation genetic strain laboratory mouse model design /development molecular cloning mutant nucleic acid hybridization protein purification protein structure function virulence

项目摘要

The broad, long-term objectives of the studies presented in this application are to define the contributions of Campylobacter jejuni potential virulence factors to disease. C. jejuni is a frequent cause of human diarrheal disease. Different C. jejuni isolates have been shown to possess various combinations of potential virulence factors: an enterotoxin, several apparently different cytotoxins, and invasiveness. None of these potential virulence properties are well-defined. The specific aims of the research proposed here are to characterize two of the cytotoxins produced by C. jejuni and to examine their role in disease. Some strains of C. jejuni produce a Shiga-like toxin (SLT). DNA isolated from C. jejuni strains hybridizes to an E. coli SLT oligonucleotide probe. Experiments designed to clone the SLT genes from a C. jejuni strain are described. The SLT genes will be cloned into E. coli and will be examined by subcloning and sequencing. Methods of expressing the genes in E. coli using expression vectors will be tried. A mutant in the cytotoxin gene will be made and returned to C. jejuni so that isogeneic mutant and parental strains can be tested in an animal model and thereby define the role of this SLT in C. jejuni disease. Also, a strain will be made that produces enough SLT to make purification feasible. This toxin can then be reliably characterized and compared to other C. jejuni cytotoxins. A recently described type of cytotoxin that caused cultured cells to become distended prior to killing them is also produced by many C. jejuni strains. Since this toxin, called cytolethal distending toxin, (CLDT), appears to be produced in quantities that will allow purification, experiments are proposed for purifying CLDT and subsequently characterizing it. Other experiments outlining methods for cloning the CLDT gene(s) are proposed. Mutants in the cloned CLDT gene(s) will be made and returned to a parental C. jejuni strain which will then be tested, along with the parental strain, in an animal model. Hybridization studies are also proposed to test the prevalence and homogeneity of CLDT genes in other C. jejuni strains and in related species.

本研究提出的广泛、长期目标应用程序是为了定义空肠弯曲杆菌的贡献疾病的潜在毒力因素。空肠弯曲杆菌是以下疾病的常见原因：人类腹泻病。不同的空肠弯曲菌分离株已被证明具有潜在毒力因子的各种组合：肠毒素、几种明显不同的细胞毒素和侵袭性。这些潜在的毒力特性都没有明确定义。这这里提出的研究的具体目标是描述其中的两个特征空肠弯曲菌产生的细胞毒素并检查它们在疾病中的作用。空肠弯曲菌的某些菌株会产生志贺样毒素 (SLT)。分离的 DNA 来自空肠弯曲菌菌株的菌株与大肠杆菌 SLT 寡核苷酸探针杂交。旨在从空肠弯曲菌菌株中克隆 SLT 基因的实验是描述的。 SLT 基因将被克隆到大肠杆菌中并进行检查通过亚克隆和测序。在大肠杆菌中表达基因的方法将尝试使用表达载体。细胞毒素基因突变将被制造并返回到空肠弯曲菌，以便同基因突变体和亲本菌株可以在动物模型中进行测试，从而确定该 SLT 在空肠弯曲菌疾病中的作用。此外，还会产生应变产生足够的 SLT 以使纯化可行。这种毒素可以可靠地表征并与其他空肠弯曲菌细胞毒素进行比较。最近描述的一种细胞毒素，可导致培养的细胞变成许多空肠弯曲菌菌株也会在杀死它们之前产生膨胀。由于这种毒素被称为细胞致死膨胀毒素 (CLDT)，似乎是生产的数量可以进行纯化，实验是建议纯化 CLDT 并随后对其进行表征。其他提出了概述克隆 CLDT 基因方法的实验。克隆的 CLDT 基因中的突变体将被制造并返回给亲本然后将与亲本菌株一起测试空肠弯曲杆菌菌株，在动物模型中。还建议进行杂交研究来测试 CLDT 基因在其他空肠弯曲菌菌株和相关物种。