From combined biochemical, ultrastructural and molecular studies conducted during the PI's FIRST AWARD, a novel model has emerged from the molecular architecture of Treponema pallidum, the etiologic agent of venereal syphilis. According to this new model, the T. pallidum outer membrane contains a rare transmembrane protein, tentatively identified as a 32-Kda polypeptide, while the major membrane immunogens (formerly thought to be located in the outer membrane) are integral cytoplasmic membrane proteins. To accommodate the newly discovered lipoprotein structures of the major membrane immunogens, the model further proposes that these immunogens comprise a """"""""family"""""""" of hydrophilic polypeptides located within the periplasmic space and anchored to the external leaflet of the cytoplasmic membrane via lipids bound to their N-terminal cysteine residues. Furthermore, we have identified potent immunostimulatory properties of these membrane immunogens (cytokine production and Ia expression by macrophages), which may derive from their lipoprotein structures. The research in this proposal will refine the above model and delineate the structural and biochemical requirements for macrophage activation by the lipoproteins. First, we will confirm that the 32-Kda protein in isolated outer membrane preparations is a rare outer membrane protein of T. pallidum (Specific Aim I). This will be accomplished by raising murine monoclonal antibodies against the rare protein and demonstrating, by a variety of techniques, including freeze-fracture cytochemistry, that they react with the surface of intact T. pallidum. Second, we will confirm the membrane topologies predicted for the lipoprotein immunogens by analyzing the native lipoproteins in T. pallidum, the cloned immunogens expressed in E. coli, and the purified lipoproteins reconstituted into liposomes (Specific Aim II). Lastly, we will characterize further the immunological properties of the lipoproteins and determine the structural features of these molecules (e.g. the covalently bound lipids) which contribution to their immunostimulatory activities (Specific Aim III). The ultimate strength of this proposal is that it presents the first coherent model for T. pallidum ultrastructure that accounts for the remarkable immunological evasiveness of the pathogen and the intense immunological phenomena which characterize human syphilis.
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