Evidence that immunization with TP0751, a bipartiteTreponema pallidumlipoprotein with an intrinsically disordered region and lipocalin fold, fails to protect in the rabbit model of experimental syphilis

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of theTreponema pallidumouter membrane (OM). TheT.pallidumOM contains a low density of integral OM proteins, while the spirochete's many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751.In silicodocking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded beta-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to otherT.pallidumlipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake ofT.pallidumby rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge withT.pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location. Author summary Development of a syphilis vaccine requires knowledge of the molecular architecture of theTreponema pallidumouter membrane (OM). TheT.pallidumOM contains a paucity of integral OM proteins, while the spirochete's lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed adhesin/protease and protective antigen. The recent increase in calycin/lipocalin structures prompted a comprehensive re-examination of TP0751. TP0751 contains two domains-an N-terminal intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin. Query of the structural databases identified related bacterial lipocalins, none known surface molecules.In silicodocking studies predicted that TP0751 binds diverse ligands along the rim of its eight-stranded beta-barrel and this was confirmed in heme binding assays. TP0751 is poorly expressed compared to otherT.pallidumlipoproteins and barely elicits antibodies in syphilis immune rabbits or patients with secondary syphilis. TP0751 antibodies did not promote uptake of spirochetes by rabbit peritoneal macrophages, and immunization of rabbits with intact, full-length TP0751 did not prevent local or disseminated infection. Our data indicate that TP0751 resides within the periplasmic space ofT.pallidumwhere it binds small molecules; we propose that the IDR facilitates ligand binding by and offloading from the lipocalin domain.