Past study of immunity in experimental syphilis has been limited by the fact that the rare outer membrane spanning proteins (TROMP) of Treponema pallidum had not been defined. This proposal is based on isolation of the T. pallidum outer membrane without the use of detergents, demonstration of outer membrane vesicle (OMV) porin activity, and definition of OMV associated protein. Proteins of 31 and 65 kDa are minor constituents of T. pallidum, but are vastly enriched in OMV. Porin activity observed in collaboration with R.B.W. Hancock resides in one of these proteins based on the known sensitivity of the black lipid bilayer assay and the protein content of the OMV. The genes encoding the 31 and the 65kDa proteins, designated tromp1 and tromp2, respectively, will be sequenced, and are expected to have architecture typical of OM proteins of gram negative bacteria, just as we found in the case of OmpL1 of Leptospira alstoni, the first OM spanning protein to be described for a pathogenic spirochete. Recombinant tromp1 and 2 (r-tromps), and cyclic peptides corresponding to surface exposed segments of these proteins will be used where appropriate in tests of porin function, proteoliposome incorporation, and correspondence to native T. pallidum OM protein epitopes. Larger scale purification of native i: pallidum OMV will be considerably more time consuming than cloning, sequencing and expression of r-tromps, but is planned because native OM protein conformation could be essential to porin function, ability to span the OM, adhesion to host molecules, and to protective immunity. With the same large-scale OM preparation we will determine if there are even less abundant proteins than the 31 and 65kDa proteins which span the OM. We will also examine the phospholipid composition of the OM. The specific adherence properties of OMV and proteoliposomal r-tromps will be tested. It is likely that there is a critical relationship between TROMP conformation and protective immunity. Therefore, native outer membrane vesicles as well as recombinant THOMP and cyclic peptides representing surface exposed TROMP epitopes will be used in parallel to immunize rabbits for determination of protective immunity. Modes of immunization will include proteoliposomes, immune stimulatory complexes (iscoms), E. coli OMV with surface exposed TROMP epitopes, and use of a novel Listeria monoytogenes- TROMP vaccine strain.
