Genetic Analysis of Cholera Toxin Structure and Function

描述(申请人提供)：每年约有40亿腹泻病例发生，占全球所有死亡病例的4%。霍乱弧菌引起的霍乱和产肠毒素大肠杆菌(ETEC)引起的水样腹泻在世界范围内造成了相当大的发病率和死亡率，ETEC占发展中国家腹泻病例的20%。仅通过公共卫生和个人卫生措施在发展中国家控制霍乱和ETEC腹泻的障碍是巨大的。美国没有批准针对霍乱或ETEC腹泻的疫苗，其他地方使用的霍乱疫苗也有很大的局限性。霍乱毒素(CT)和与之密切相关的I型不耐热肠毒素(LTI)引起水样腹泻，分别是霍乱弧菌和ETEC的主要毒力因子。霍乱弧菌和ETEC产生无关的菌毛，这些菌毛在它们在小肠的定植中起着至关重要的作用，而针对特定菌毛成分的抗体可以阻止定植和预防腹泻。抗CT或抗LTI抗体单独不能预防腹泻，但据信它们与阻止定植的抗体协同作用，分别加强对霍乱和ETEC腹泻的保护。我们的长期目标是：1)在分子水平上了解CT和LTI的功能，2)开发针对人类霍乱弧菌和ETEC感染的安全有效的疫苗。在下一个项目期间，我们将继续研究霍乱毒素结构和功能的基本方面。我们将研究具有催化活性的CTA1片段是如何识别其生理底物GSA的，哪些因素决定了CTA1与其变构激活剂ADP核糖化因子6(ARF6)相互作用的功能后果，以及五聚体B亚单位与神经节苷脂GM1受体的单价和多价结合如何影响CT与靶细胞的相互作用。我们最近开发了新的方法来制作基于CT或LT的肠毒素-粘附素嵌合体，并证明了这种嵌合体含有来自ETEC的毛尖粘附素蛋白的强大的粘膜免疫原性。在下一个项目期间，我们将制造和测试各种肠毒素-粘附素嵌合体作为霍乱和ETEC腹泻的候选疫苗，并进行初步实验，将这些技术应用于开发肠毒素-粘附素嵌合体作为预防大肠杆菌和奇异变形杆菌引起的尿路感染的候选疫苗。我们提出的肠毒素-粘附素嵌合体作为候选疫苗的研究代表了几十年来获得的关于肠毒素结构和功能的基本知识的应用，用于开发有效的疫苗来对抗肠道和尿路的细菌感染，这是主要的世界卫生问题。

DESCRIPTION (provided by applicant): About 4 billion cases of diarrhea occur annually and cause 4% of all deaths worldwide. Cholera caused by Vibrio cholerae and watery diarrhea caused by enterotoxigenic Escherichia coli (ETEC) cause substantial morbidity and mortality worldwide, and ETEC accounts for up to 20% of diarrhea cases in developing countries. The obstacles to controlling cholera and ETEC diarrhea in developing countries by public health and personal sanitation measures alone are formidable. No vaccine against cholera or ETEC diarrhea is approved in the United States, and cholera vaccines used elsewhere have significant limitations. Cholera toxin (CT) and the closely related type I heat-labile enterotoxin (LTI) cause watery diarrhea and are major virulence factors of V. cholerae and ETEC, respectively. V. cholerae and ETEC produce unrelated pili that have essential roles in their colonization of the small intestine, and antibodies against specific pilus components can block colonization and prevent diarrhea. Anti-CT or anti-LTI antibodies alone do not prevent diarrhea, but they are believed to act synergistically with antibodies that block colonization to provide enhanced protection against cholera and ETEC diarrhea, respectively. Our long-term goals are to: 1) understand at the molecular level the functions of CT and LTI, and 2) to develop safe and effective vaccines against V. cholerae and ETEC infections of humans. During the next project period, we will continue to study fundamental aspects of cholera toxin structure and function. We will investigate how the catalytically active CTA1 fragment recognizes its physiological substrate Gsa, what factors determine the functional consequences of interactions between CTA1 and its allosteric activator ADP ribosylation factor 6 (ARF6), and how monovalent vs. multivalent binding of ganglioside GM1 receptors by the pentameric B subunit affects the interactions of CT with target cells. We recently developed novel methods to make CT-based or LT-based enterotoxin-adhesin chimeras and demonstrated potent mucosal immunogenicity of such chimeras containing pilus tip adhesin proteins from ETEC. During the next project period, we will make and test a variety of enterotoxin-adhesin chimeras as candidate vaccines against cholera and ETEC diarrhea, and we will perform preliminary experiments to apply these technologies for the development of enterotoxin-adhesin chimeras as candidate vaccines against urinary tract infections caused by Escherichia coli and Proteus mirabilis. Our proposed studies on enterotoxin-adhesin chimeras as vaccine candidates represent applications of fundamental knowledge about enterotoxin structure and function that has been acquired over several decades for development of effective vaccines against bacterial infections of the intestinal tract and the urinary tract that are major world health problems.