There has been recent progress in understanding the molecular basis of two key aspects of the immunobiology of Treponema pallidum, the antigenic inertness of its surface, and its resistance to killing by immune mechanisms. Freeze-fracture electron microscopy has shown that the outer membrane of T. pallidum contains approximately two orders of magnitude less transmembrane protein than that of gram negative bacteria. We have learned that this rare outer membrane protein, which has been given the acronym, TROMP, can be aggregated by antibodies from rabbits immune to syphilitic infection. Such antibody mediated TROMP aggregation appears to be a prerequisite for efficient complement activation and killing of T. pallidum. The primary focus of this proposal is the identification and characterization of TROMP. The fragility of T. pallidum, the absence of a genetic exchange system for spirochetes, and the rarity of TROMP are considerations in determining its identity definitively. We have developed a surrogate genetic system using a cloning vector which facilitates construction of fusions between the signal sequences of T. pallidum genes and an alkaline phosphatase gene from which the signal sequence has been removed. This phoA vector, pMG1, has been used to express T. pallidum-phoA fusion proteins which correspond to the genes which might be identifiable by TnphoA insertion were it possible to use this transposon in T.pallidum. It is possible to identify those T. pallidum-alkaline phosphatase fusion proteins which remain anchored to the cytoplasmic membrane because they have noncleavable signal sequences. The fusion proteins with cleavable signal sequences can be grouped into those which are acylated by E. coli and correspond to T. pallidum lipoproteins, those which correspond to T. pallidum periplasmic proteins. and those with membrane spanning sequences, which will be regarded as TROMP candidates. Rabbits will be immunized with cloned TROMP candidates. The resulting antisera will be used in an assay for demonstrating antibody to TROMP by freeze-fracture electron microscopy, for in vitro treponemicidal assays, and for in vitro assays which measure the ability of T. pallidum to adhere to cells, to fibronectin, and to invade the intercellular junctions of endothelial monolayers. The results of these in vitro studies will be correlated with the outcomes of intradermal challenge experiments with the immunized rabbits. To test the idea that antibodies with specificity for subsurface targets, such as the cytoplasmic membrane, may enhance the efficiency of killing, we will identify two relatively abundant proteins of the cytoplasmic membrane which have multiple membrane spanning sequences and study these proteins in conjunction with the TROMP candidates.
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