PROBING THE MECHANISM OF COMPLEMENT-INDEPENDENT BACTERICIDAL ANTIBODIES

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： 我们先前已经证明，通过使用单链可变片段(ScFv)，针对疏螺旋体的补体非依赖性抗体的杀菌作用驻留在抗体可变区(LaRocca等人)。2007年。J.免疫。在印刷中)。我们目前的工作集中在这种补体非依赖性抗体的杀菌作用机制上。我们知道，这些抗体直接作用于疏螺旋体的外膜，当疏螺旋体在葡聚糖等大糖存在的情况下暴露于抗体时，观察到外膜的保护作用。我们认为这表明外膜是由于渗透溶解和孔形成而被破坏的。对于较小的糖，我们观察到了对外膜的部分保护。换句话说，较小的糖最初会保护外膜免受溶解，但最终会失去这种保护能力。我们认为，这表明外膜孔随着时间的推移而产生和增大，类似于抗菌肽利用的膜解体机制。我们一直观察到这些抗体引起的细胞膜起泡，并认为这与毛孔的形成和毛孔大小的增加有关。我们有兴趣利用RVBC设备在葡聚糖存在的情况下观察抗体引起的外膜毛孔。根据渗透保护实验，孔径估计在4.4到12 nm之间，但可以增大到需要分子直径28 nm的糖来进行渗透保护。 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，细胞被急速冷冻。对对照细胞以及抗体处理后不同时间段的细胞(共6个实验者)进行整体冷光显微镜观察。即使在最短的时间内，也可以看到大量的起泡和外膜的小破裂。

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.