Functional analysis of Helicobacter pylori VacA toxin

幽门螺杆菌VacA毒素的功能分析

• 批准号: 6737639
• 负责人: Victor J. Torres
• 金额: $ 3.59万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-01 至 2006-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6737639
• 关键词: Helicobacter; SDS polyacrylamide gel electrophoresis; aminoacid; bacterial cytopathogenic effect; bacterial toxins; cytotoxicity; enzyme linked immunosorbent assay; gene deletion mutation; genetic library; host organism interaction; phenotype; predoctoral investigator; protein structure function; virulence; virus assembly; virus protein; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Helicobacter pylori infection is a risk factor for duodenal ulcer disease and gastric cancer. One H. pylori virulence factor associated with disease is the vacuolating cytotoxin (VacA). VacA exerts a variety of effects on epithelial cells in vitro, including the formation of intracellular vacuoles and anion-selective pores in the plasma membrane. VacA domains involved in assembly of the toxin into oligomeric structures or membrane channels have not been analyzed yet. Also, domains of VacA essential for toxin binding to cells have not yet been analyzed in any detail. The aims of this proposal are: (i) identification of domain(s) required for VacA assembly into oligomeric structures. To accomplish this aim, the yeast-two hybrid method is being used. Preliminary data suggest that the yeast-two hybrid system is suitable for the screening of oligomerization domains within VacA. (ii) mapping the cell binding domain(s) required for VacA binding to target cells. To achieve this aim, a library of VacA carboxyl-terminal deletions has been generated. The mutant recombinant toxins are being expressed in E. coli and tested for their cytotoxic activity as well as their ability to bind to cells. (iii) understanding the mechanism of the dominant-negative phenotype observed with the VacA delta6-27 deletion mutant. To fulfill this aim I am using the VacA recombinant system to determine the minimum amino acid sequence required for the delta6-27-VacA toxin to exhibit a dominant-negative phenotype.

描述（由申请方提供）：幽门螺杆菌感染是十二指肠溃疡疾病和胃癌的危险因素。 一个H与疾病相关的幽门螺杆菌毒力因子是空泡细胞毒素（VacA）。 VacA在体外对上皮细胞产生多种作用，包括形成细胞内空泡和质膜上的阴离子选择性孔。 参与毒素组装成寡聚体结构或膜通道的VacA结构域尚未被分析。 此外，尚未对毒素结合细胞所必需的VacA结构域进行任何详细分析。 该提议的目的是：（i）鉴定VacA组装成寡聚体结构所需的结构域。 为了实现这一目标，正在使用酵母双杂交方法。 初步数据表明，酵母双杂交系统是适合于筛选寡聚化结构域内的VacA。 (ii)定位VacA结合靶细胞所需的细胞结合结构域。 为了实现这一目标，已经产生了VacA羧基末端缺失的文库。 突变重组毒素在E.大肠杆菌中，并测试其细胞毒性活性以及它们与细胞结合的能力。 (iii)理解VacA δ 6 -27缺失突变体观察到的显性阴性表型的机制。 为了实现这一目标，我正在使用VacA重组系统来确定δ 6 -27-VacA毒素表现出显性阴性表型所需的最小氨基酸序列。