CHARACTERIZATION OF THE PHYSIOLOGICAL REQUIREMENTS FOR THE BACTERICIDAL EFFECTS OF A MONOCLONAL-ANTIBODY TO OSPB OF BORRELIA-BURGDORFERI BYCONFOCAL MICROSCOPY

A confocal microscopy study was undertaken to characterize the bacteri cidal effects of the Fab fragments of CB2, an immunoglobulin G1 kappa murine monoclonal antibody, to an epitope in the carboxy region of the outer surface protein B (OspB) of Borrelia burgdorferi. Simultaneous direct labeling of both fixed and live spirochetes with fluorochrome-l abeled Fab-CB2 and 11G1, and an immunoglobulin M kappa monoclonal anti body to OspA, showed that OspA and OspB seem to colocalize in dead spi rochetes but do not appear to be physically associated when the organi sms are alive. A polar blob composed of a Fab-CB2-OspB complex, follow ed by incorporation of 11G1-OspA, precedes the formation of a spheropl ast, The spheroplasts contain both OspA and OspB and are a terminal st age in the bactericidal process induced by Fab-CB2. Outer membrane des tabilization by Fab-CB2, but not cell wall or cytoplasmic membrane alt erations, was demonstrated experimentally by the sequential treatment of spirochetes with Fab-CB2 and monoclonal antibodies to flagellin and DnaK. The action of Fab-CB2 is epitope specific, as another monoclona l antibody to an epitope in the amino terminus of OspB was not bacteri cidal, The bactericidal effect of Fab-CB2 is not dependent on the indu ction of spirochetal proteases but is dependent on the presence of Ca2 + and Mg2+, Supplementation of Ca2+- and Mg2+-free medium with these c ations restored the bactericidal effects of Fab-CB2. The mechanism by which a Fab fragment of an antibody destroys a bacterium directly may represent a novel form of antibody-organism interaction.