Bordetella adenylate cyclase toxin: the role of cell interaction in toxin functio

描述(申请人提供)：腺苷环化酶毒素(AC毒素)是由百日咳波尔德氏菌引起的疾病所必需的，这种疾病在过去20年里再次出现在美国。AC毒素由八种波尔德氏菌中的七种表达，波尔德氏菌会导致人类和一系列动物的呼吸道疾病。该毒素分子是腺苷环化酶和与毒素中重复序列(RTX)家族同源的结合域的独特杂交。AC毒素通过与细胞膜结合并将其催化结构域跨脂双层移位而中毒宿主细胞，导致细胞内cAMP的无调控生成。通过针对催化域的抗体或某些突变来防止易位，将增强毒素的另一个主要功能，即形成导致红细胞溶解的寡聚孔，并有助于巨噬细胞的非凋亡性细胞死亡。中毒程度与中性粒细胞和巨噬细胞表达最丰富的整合素受体CD11b/CD18的相对表面表达有关，但AC毒素与CD11b/CD18的关系尚不清楚。我们的初步数据表明，CD11b/CD18不仅增加了细胞对中毒的敏感性，而且，当高浓度的毒素作用于细胞时，实际上限制了中毒，同时可能促进低聚物的形成。我们将探讨AC毒素与CD11b/CD18相互作用的基本机制。毒素结合和功能的决定因素可能可以解释一个新的发现：上皮单层的细胞对施加在顶端表面的AC毒素中毒不敏感，令人惊讶的是，这种毒素可以中毒迄今测试的所有细胞类型，而且重要的是因为细菌在感染时首先遇到这些细胞。这些研究将有助于阐明细菌毒素形成毛孔的机制，以及由所有波氏杆菌引起的疾病的发病机制。这个拟议的项目将延续我从19岁开始对基础科学的兴趣。我的导师埃里克·休利特博士拥有超过25年的研究百日咳杆菌的经验，他是细菌毒素领域的杰出科学家。他对通过科学合作学习的强调将补充我有组织的课程工作计划。K08奖，加上我的导师和弗吉尼亚大学的资源，将为我作为临床科学家的全面发展提供必要的支持。 相关：AC毒素由八种波尔德氏菌中的七种表达，也是由其他致病细菌产生的RTX成孔蛋白毒素家族的成员，如泌尿系致病性大肠杆菌。在这里，我们将描述AC毒素与细胞结合的基本机制，并说明这些机制影响毒素的功能，并确定其作为毒力因子的作用。

DESCRIPTION (provided by applicant): The adenylate cyclase toxin (AC toxin) is necessary for disease caused by Bordetella pertussis, which has reemerged in the United States over the last two decades. AC toxin is expressed by seven of eight species of Bordetella which cause respiratory disease in humans and a range of animals. The toxin molecule is a unique hybrid of an adenylate cyclase enzyme and a binding domain homologous to the repeats-in-toxin (RTX) family of pore-forming bacterial protein toxins. AC toxin intoxicates host cells by binding to the cell membrane and translocating its catalytic domain across the lipid-bilayer, resulting in unregulated generation of intracellular cAMP. Prevention of translocation by an antibody to the catalytic domain or by certain mutations will enhance the other major function of the toxin, formation of oligomeric pores which cause lysis of erythrocytes and contribute to non-apoptotic cell death of macrophages. The magnitude of intoxication correlates with the relative surface expression of the (32 integrin, CD11b/CD18, a receptor expressed most abundantly on neutrophils and macrophages; however, the relationship between AC toxin and CD11b/CD18 is not well defined. Our preliminary data show that CD11b/CD18 does not simply increase sensitivity of cells to intoxication, but, when high concentrations of toxin are applied to cells, actually limits intoxication while possibly enhancing oligomer formation. We will investigate the basic mechanisms of the interaction between AC toxin and CD11 b/CD18. The determinants of toxin binding and function may explain a novel finding: cells of an epithelial monolayer are insensitive to intoxication by AC toxin applied to the apical surface, surprising because the toxin can intoxicate all cell types so far tested, and important because the bacterium first encounters these cells upon infection. These studies will shed light upon the mechanism of pore formation by bacterial toxins, and the pathogenesis of disease caused by all Bordetellae. The proposed project will continue my interest in basic science that began at the age of 19. My mentor, Dr. Erik Hewlett, with over 25 years of experience" studying Bordetella pertussis, is a preeminent scientist in the field of bacterial toxins. His emphasis on learning through scientific collaboration will complement my plan for structured coursework. The K08 award, in combination with the resources of my mentor and the University of Virginia, will provide me with the support necessary for my full development as a clinician scientist. RELEVANCE: AC toxin is expressed by seven of the eight species of Bordetella and is also a member of the RTX family of pore-forming bacterial protein toxins that are produced by other pathogenic bacteria such as uropathogenic Escherichia coli. Here, we will characterize the basic mechanisms of cell binding by AC toxin and show that these mechanisms affect the function of the toxin and determine its role as a virulence factor.