To study the role of the rare T. pallidum (Tp) outer membrane (OM) spanning proteins in pathogenesis and immunity, we isolated Tp OM vesicles (OMV). OMV porin activity resides in a 31 kDa protein, Tromb1. Native Tromp1 is 31, 510 Da, and is processed from a 33,571 Da precursor. While native Tromp1 is hydrophobic, and trimeric, urea creates a hydrophilic monomer, indicating that the hydrophobicity of the native protein is conformationally determined. Monomeric rTromp1 has no poring activity and is hydrophilic. Renatured rTromp1 is trimeric and hydrophobic, with porin activity like native Tromp1. Renatured rTromp1 formed intra membranous particles in proteoliposomes. OMV were used to generated mouse antiserum that showed a 100% killing endpoint titer 32 times greater than time of immune rabbit serum (IRS). The OMV ant- serum bound Tromp1, Tromp2, and four lipoproteins were fully removed, while Tromp antibodies remained. The adsorbed serum showed no reduction in its high-titered treponemicidal activity, suggesting that Tromp1 and/or Tromp2 are the targets of this activity. Treponemicidal activity greater than that found in IRS has never previously been demonstrated.
Our specific aims are therefore: 1. Determine the significance and basis of OMV induced treponemicidal antibodies. We will learn if OMV can convey protection in experimental syphilis. The possibility that treponemicidal antibodies. We will learn if OMV can covey protection in experimental syphilis. The possibility that there are relevant OMV proteins other than Tromp1 and 2 will be rigorously considered. The role of OMV in pathogenesis will be addressed by use of adherence and invasion assays. 2. Determine the role of Tromp1 in pathogenesis and immunity. Mass spectrometry adapted to nanogram amounts will be used to insure that rTromp1 faithfully duplicates the primary structure of native Tromp1. The ability of renatured rTromp1 and native Tromp1 to induce treponemicidal antibodies and protective immunity will be assessed, along with the role of Tromp1 in pathogenesis. 3. Determine the role of Tromp2 in pathogenesis and immunity. Tromp2 has conformationally determined hydrophobicity, but lacks porin activity. Tromp2 is considerably less abundant than Tromp1. Definitive determination of the primary structure of mature Tromp2 will be used as the basis for creating a rTromp2 whose primary structure is identical to the native protein. rTromp2 will be renatured and studied as outlined for rTromp1.
