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.
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