ULCER-ASSOCIATED MAJOR ANTIGEN OF HELICOBACTER PYLORI

幽门螺杆菌溃疡相关主要抗原

• 批准号: 2749521
• 负责人: MURALI K TUMMURU
• 金额: $ 10.87万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749521
• 关键词: Helicobacter; SDS polyacrylamide gel electrophoresis; bacterial antigens; bacterial genetics; biopsy; cell adhesion; cytokine; duodenal ulcer; endoscopy; enzyme linked immunosorbent assay; humoral immunity; immunoglobulin A; inflammation; interleukin 1; interleukin 8; leukocyte activation disorder; microorganism culture; molecular pathology; peptic ulcer; plasmids; protein purification; site directed mutagenesis; virulence

Helicobacter pylori is a curved, microaerophilic, gram-negative bacterium that is the major cause of chronic gastritis in humans. While most infected persons remain asymptomatic, infection with this pathogen is a significant risk factor for the development of peptic ulcer disease and gastric carcinoma. The major objective of the proposed work is to characterize the role of the 131 kDa immunodominant antigen (CagA; cytotoxin-associated gene product) in the pathogenesis of H.pylori- associated duodenal ulcer disease. About 50-60% of H.pylori isolates produce the CagA antigen, and virtually all H. pylori-infected patients with duodenal ulceration develop a serologic response to CagA. Previous studies have shown that mucosal neutrophil infiltration and epithelial surface degeneration are significantly greater in patients with a local gastric immune response to CagA than in those without such a response. Previous studies have also shown that CagA-producing H. pylori strains induce significantly more interleukin-8 (lL-8) synthesis by gastric epithelial cells than do CagA-negative strains. In addition,recent studies have shown that patients infected with CagA+ strains induce a specific pattern of cytokine expression in gastric tissue involving IL-1 alpha and IL-1 beta. The gene encoding CagA has now been cloned, and recombinant CagA protein is recognized by sera from an H.pylori-infected patients. H.pylori can vary the size of CagA and the size variation is associated with deletion or insertion of repeat sequences. Sequence analysis of the region upstream from cagA, which is not present in CagA strains, identified three genes (cagB, cagC, and cagD ). The deduced CagC sequence was found to have significant homology with the Bordetella pertussis toxin secretion protein (PtIC). The hypothesis of this study is that the CagA antigen of H.pylori is associated with duodenal ulcer disease, and is an important virulence factor. The specific aims are 1) to study the relationship between CagA production and H.pylori-associated duodenal ulcer disease, 2) to determine the role of CagA in gastric inflammation, 3) to characterize the genetic differences between CagA+ and CagA H.pylori strains.To accomplish the first objective, native CagA from H.pylori strain 84-183 will be purified and its structural and biochemical characteristics determined. Next, mucosal immune responses to CagA will be compared in patients with gastritis alone and those with duodenal ulceration. Subsequently, H.pylori isolates from patients with gastritis alone and duodenal ulcer will be tested for CagA size and determine the relationship between the type of CagA expressed and clinical outcome. Isogenic pairs of CagA+ and CagA strains will be used to determine whether CagA plays a role in cytokine induction and neutrophil activation within gastric tissues. The cagA neighboring genes that are present exclusively in CagA+ strains will be characterized and disrupted by insertional mutagenesis, and the mutants will be tested for cytotoxin secretion and/or cytokine induction. Understanding the pathogenic role of CagA and its neighboring gene products in H.pylori infection may aid in the development of strategies to decrease the morbidity associated with complications of H.pylori infection.

幽门螺杆菌是一种弯曲、微需氧、革兰氏阴性细菌 这是人类慢性胃炎的主要原因。虽然大多数 感染者仍然无症状，感染这种病原体是一种 消化性溃疡病发生的重要危险因素 胃癌。拟议工作的主要目标是 描述 131 kDa 免疫显性抗原 (CagA; 细胞毒素相关基因产物）在幽门螺杆菌发病机制中的作用 相关十二指肠溃疡病。约 50-60% 的幽门螺杆菌分离株 产生 CagA 抗原，几乎所有幽门螺杆菌感染的患者 十二指肠溃疡会对 CagA 产生血清学反应。以前的 研究表明，粘膜中性粒细胞浸润和上皮细胞浸润 局部病变患者的表面变性明显更大 胃对 CagA 的免疫反应高于没有这种反应的胃。 先前的研究还表明，产生 CagA 的幽门螺杆菌菌株 显着诱导更多的胃白细胞介素 8 (IL-8) 合成 上皮细胞比 CagA 阴性菌株更有效。此外，最近的研究 研究表明，感染 CagA+ 菌株的患者会诱导特定的 胃组织中涉及IL-1α和的细胞因子表达模式 IL-1 β。编码CagA的基因现已被克隆并重组 CagA 蛋白可被幽门螺杆菌感染患者的血清识别。 幽门螺杆菌可以改变 CagA 的大小，并且大小变化与之相关 删除或插入重复序列。 序列分析 cagA 上游区域，该区域在 CagA 菌株中不存在， 鉴定了三个基因（cagB、cagC 和 cagD）。推导的CagC序列 被发现与百日咳博德特氏菌毒素具有显着的同源性 分泌蛋白（PtIC）。本研究的假设是 CagA 幽门螺杆菌抗原与十二指肠溃疡病相关，是一种 重要的毒力因子。具体目标是 1) 研究 CagA 产生与 H.pylori 相关十二指肠的关系 溃疡病，2)确定CagA在胃炎症中的作用， 3) 表征CagA+和CagA H.pylori之间的遗传差异 为了实现第一个目标，来自幽门螺杆菌的天然 CagA 菌株84-183将被纯化，其结构和生化 特性确定。接下来，对 CagA 的粘膜免疫反应将是 单纯胃炎患者与十二指肠胃炎患者的比较 溃疡。随后，从胃炎患者体内分离出幽门螺杆菌 单独和十二指肠溃疡将测试 CagA 大小并确定 CagA 表达类型与临床结果之间的关系。 CagA+ 和 CagA 菌株的同基因对将用于确定是否 CagA 在细胞因子诱导和中性粒细胞激活中发挥作用 胃组织。专门存在的 cagA 邻近基因 在 CagA+ 菌株中，将通过插入来表征和破坏 诱变，并且将测试突变体的细胞毒素分泌和/或 细胞因子诱导。了解 CagA 及其致病作用 幽门螺杆菌感染中的邻近基因产物可能有助于发育 降低并发症相关发病率的策略 幽门螺杆菌感染。