MECHANISMS OF SECRETORY IGA MEDIATED IMMUNITY TO ENTERIC PATHOGENS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 摘要： 分泌型IgA(SIgA)抗体覆盖在胃肠道粘膜表面，是抵御革兰氏阴性肠道病原体的第一道防线，包括大肠杆菌、福氏志贺氏菌、霍乱弧菌和鼠伤寒沙门氏菌。一般认为，SIgA主要通过结合细菌脂多糖和促进细菌凝集来保护粘膜表面，从而防止细菌与上皮表面的接触。然而，我们实验室最近与William Samsonoff博士和Richard Cole博士合作的研究表明，SIgA抗体通过促进细菌外膜的变化直接干预细菌的发病，细菌外膜通过专门的分泌装置阻止鞭毛旋转和阻止分泌。具体地说，我们认为对抗内毒素的SIgA导致外膜局部扭曲，导致机械敏感毛孔打开，随后质子动力耗散，这是已知的驱动鞭毛旋转和分泌的动力。我们建议使用冷冻电子断层扫描，在没有和存在抗内毒素单抗IgA抗体Sal4的情况下，对鼠伤寒沙门氏菌的外膜进行可视化。在初步研究中，我们优化了阻止细菌运动和上皮细胞入侵所需的抗体和细菌浓度。基于Manella博士提出的工作，我们预计冷冻电子断层扫描将使我们能够更详细地显示细菌鞭毛、分泌装置和外膜结构。 在前一报告期，对照细胞、抗体处理细胞和具有外膜突变的细胞被急速冷冻。当用抗体处理时，整个细菌的冷冻-EM图像显示出一层厚厚的模糊的外壳。当用抗体处理时，突变的细胞显示出更薄的外套。目前正在对数据进行解释，并计划进行进一步的实验。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Secretory IgA (SIgA) antibodies coat the mucosal surfaces of the gastrointestinal tract and serve as the first line of defense against Gram negative enteric pathogens, including Escherichia coli, Shigella flexneri, Vibrio cholerae, and Salmonella typhimurium. It is generally assumed that SIgA protects mucosal surfaces primarily by binding bacterial lipopolysaccharide and promoting bacterial agglutination, thereby, preventing bacterial contact with epithelial surfaces. However, recent studies from our laboratory in collaboration with Drs. William Samsonoff and Richard Cole suggest that SIgA antibodies directly interfere with bacterial pathogenesis by promoting changes in the bacterial outer membrane that arrest flagella rotation and block secretion through specialized secretion apparatuses. Specifically, we propose that SIgA against LPS causes localized distortions of the outer membrane that result in the opening of mechanosensitive pores and the subsequent dissipation of the proton motive force, which is know to drive both flagella rotation and secretion. We propose to use cryoelectron tomography to visualize the outer membrane of Salmonella typhimurium in the absence and presence of the anti-LPS monoclonal IgA antibody Sal4. In preliminary studies, we have optimized antibody and bacteria concentrations required to arrest bacterial motility and invasion of epithelial cells. Based on work presented by Dr. Manella, we expect that cryoelectron tomography will enable us to visualize in great detail the bacterial flagella, secretion apparatuses and outer membrane architecture. In the previous reporting period, control cells, antibody-treated cells, and cells with an outer-membrane mutation were plunge-frozen. Cryo-EM images of whole-mount bacteria showed a thick fuzzy coat when treated with the antibody. The mutant cells showed a thinner coat when treated with the antibody. The data is being interpreted, and further experiments are planned.