Treponema pallidum, an obligate human pathogen, has an outer membrane (OM) whose physical properties, ultrastructure and composition differ markedly from those of phylogenetically distant Gram-negatives. We developed structural models for the outer membrane protein (OMP) repertoire of T. pallidum Nichols using solved Gram-negative structures, computational tools, and small angle X-ray scattering (SAXS) of selected recombinant periplasmic domains. The T. pallidum ‘OMPeome’ harbors two ‘stand-alone’ proteins (BamA and LptD) involved in OM biogenesis and four paralogous families involved in influx/efflux of small molecules: 8-stranded β-barrels, long-chain fatty acid transporters (FadLs), OM factors (OMFs) for efflux pumps, and T. pallidum repeat proteins (Tprs). BamA (TP0326), the central component of a BAM/TAM hybrid, possesses a highly flexible POTRA1-5 predicted to interact with TamB (TP0325). TP0515, an LptD ortholog, contains a novel, unstructured C-terminal domain that models inside the β-barrel. T. pallidum has four 8-stranded β-barrels, each containing positively charged extracellular loops that could contribute to pathogenesis. Three of five FadL-like orthologs have a novel α-helical, presumptively periplasmic C-terminal extension. SAXS and structural modeling further supported the bipartite membrane topology and tri-domain architecture of full-length members of the Tpr family. T. pallidum’s two efflux pumps presumably extrude noxious small molecules via four co-expressed OMFs with variably charged tunnels. For BamA, LptD and OMFs, we modeled the molecular machines that deliver their substrates into the OM or external milieu. The spirochete’s extended families of OM transporters collectively confer a broad capacity for nutrient uptake. The models also furnish a structural roadmap for vaccine development. Importance The unusual outer membrane (OM) of T. pallidum, the syphilis spirochete, is the ultrastructural basis for its well-recognized capacity for invasiveness, immune evasion, and persistence. In recent years, we have made considerable progress identifying T. pallidum’s repertoire of OMPs. Herein, we developed three-dimensional (3D) models for the T. pallidum Nichols OMPeome using structural modeling, bioinformatics, and solution scattering. The OM contains three families of OMP transporters, an OMP family involved in extrusion of noxious molecules, and two ‘stand alones’ involved in OM biogenesis. This work represents a major advance towards elucidating host-pathogen interactions during syphilis, understanding how T. pallidum, an extreme auxotroph, obtains a wide array of biomolecules from its obligate human host, and developing a vaccine with global efficacy.

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM 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 silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded β-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 other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits

Recent identification and structural modeling of Treponema pallidum ’s ( Tp ) repertoire of outer membrane proteins (OMPs) represent a critical breakthrough in the decades long quest for a syphilis vaccine. However, little is known about the antigenic nature of these β-barrel-forming OMPs and, more specifically, their surface exposed regions, the extracellular loops (ECLs).

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Background Development of a vaccine is a necessary step towards curtailing the global syphilis epidemic. Towards this end, our group has utilized bioinformatic and biophysical methods to characterize T. pallidum's repertoire of outer membrane proteins (OMPs): the two 'stand-alones', BamA and LptD, and the T. pallidum repeat (Tpr), 8-stranded b-barrel, outer membrane factor (OMF) for efflux pump, and FadL paralogous families. We have hypothesized that antibodies against extracellular loops (ECLs) of OMPs are responsible for the opsonic and protective activities of immune rabbit serum (IRS). We used Pyrococcus furiosus thioredoxin (PfTrx) as a scaffold to present ECLs for assessment of reactivity with syphilitic sera and quantitation of circulating ECL-specific Bcells. To develop this system, we focused on BamA/ECL4 which we previously have shown is immunogenic and a target for opsonic antibodies. Methods PfTrx was engineered to express BamA-ECL4 (PfTrx-BamA/ECL4) with a N-terminal His6-and C-terminal Avi-tags for Ni-NTA purification and in vivo biotinylation, respectively. Reactivity of IRS and human syphilitic sera (HSS) with PfTrx-BamA/ECL4 was assessed by immunoblotting. To determine the accessibility of the BamA/ECL4 epitope, a pull-down assay was performed by incubating PfTrx-BamA/ECL4 or PfTrx with IgG from IRS or normal rabbit serum (NRS) immobilized on protein G agarose beads. BamA/ECL4-specific IgG+ B-cells were identified by flow cytometry using PfTrx-BamA/ECL4 conjugated with streptavidin (SP)-Alexa Fluor 647 and SP-Brilliant Violet-421. PfTrx was conjugated to SP-APC-Cy7 to exclude non-specific binding to scaffold. Results IRS and HSS recognized PfTrx-BamA/ECL4 but not PfTrx. PfTrx-BamA/ECL4 was pulled down by IgG from IRS but not NRS, while PfTrx was not pulled down by either. 1.66% of circulating IgG+ B-cells were PfTrx-BamA/ECL4-specific. Conclusion When used in conjunction with OMP structural models, PfTrx-ECL is a promising platform for identification of antigenic ECLs and isolation of ECL-specific B-cells for the generation of anti-ECL monoclonal antibodies with opsonic activity.