PROBING THE MECHANISM OF COMPLEMENT-INDEPENDENT BACTERICIDAL ANTIBODIES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 摘要： 我们先前已经通过使用单链可变片段（scFv）显示针对疏螺旋体属的补体非依赖性抗体的杀菌作用存在于抗体可变区中（LaRocca等人2007. Immunol. In Press）。 我们目前的工作重点是这种类型的补体非依赖性抗体的杀菌作用的机制。 我们知道这些抗体直接作用于疏螺旋体的外膜，并且已经观察到当疏螺旋体在存在大糖（例如葡聚糖）的情况下暴露于抗体时外膜的保护。 我们认为，这表明，外膜被破坏，由于渗透溶解和孔的形成。 对于较小的糖，我们观察到外膜的部分保护。 换句话说，较小的糖最初保护外膜免受裂解，但最终失去这种保护能力。 我们认为，这表明外膜孔的产生和大小随时间的推移而增加，类似于抗微生物肽所利用的膜崩解机制。 我们一直观察到响应于这些抗体的膜起泡，并认为这与孔形成和孔径增加有关。 我们感兴趣的是利用RVBC设施可视化外膜孔所造成的抗体在葡聚糖的存在。 基于抗氧化保护实验，孔径估计为直径在4.4和12 nm之间，但尺寸可以增加到抗氧化保护需要分子直径为28 nm的糖的程度。 在上一个报告所述期间，Benach博士和LaRocca先生带着标本访问了RVBC，细胞被急冻。 对照细胞以及抗体处理后增加时间段的细胞（总共六个实验者）作为整体样品通过冷冻-EM检查。 即使在最短的时间内，也可以看到大量的水泡和外膜的小破裂。

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: We have previously shown that the bactericidal action of a complement-independent antibody against Borrelia resides in the antibody variable region through use of a single chain variable fragment (scFv) (LaRocca et al. 2007. J. Immunol. In Press). Our current work focuses on the mechanism of bactericidal action utilized by this type of complement-independent antibody. We know that these antibodies act directly at the outer membrane of Borrelia and have observed a protection of the outer membrane when the Borrelia are exposed to antibody in the presence of large sugars, such as dextran. We believe that this suggests that the outer membrane is destroyed due to osmotic lysis and pore formation. With smaller sugars we have observed partial protection of the outer membrane. In other words, smaller sugars initially protect the outer membrane from lysis but eventually lose this capacity for protection. We believe that this suggests that outer membrane pores are created and increase in size over time, similar to membrane disintegration mechanisms utilized by antimicrobial peptides. We have always observed membrane blebbing in response to these antibodies and believe that this is linked to pore formation and increasing pore size. We are interested in utilizing the RVBC facility to visualize outer membrane pores caused by the antibodies in the presence of dextran. Based on osmoprotection experiments, the pore size is estimated to be between 4.4 and 12 nm in diameter but can increase in size to the extent that a sugar of molecular diameter 28 nm is needed from osmoprotection. In the previous reporting period, Dr. Benach and Mr. LaRocca visited the RVBC with specimens, and cells were plunge-frozen. Control cells, as well as cells at increasing time periods after antibody treatment (a total of six experimentrs) were examined as whole mounts by cryo-EM. Much blebbing, and small ruptures of the outer membrane, were seen even at the shortest time period.