Structural Modeling of the Treponema pallidum Outer Membrane Protein Repertoire: a Road Map for Deconvolution of Syphilis Pathogenesis and Development of a Syphilis Vaccine

Abstract

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-negative bacteria. We developed structural models for the outer membrane protein (OMP) repertoire (OMPeome) 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 the 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 β-barrel assembly machine (BAM)/translocation and assembly module (TAM) hybrid, possesses a highly flexible polypeptide-transport-associated (POTRA) 1-5 arm 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 tridomain architecture of full-length members of the Tpr family. T. pallidum's two efflux pumps presumably extrude noxious small molecules via four coexpressed 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 road map 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 in identifying T. pallidum's repertoire of OMPs. Here, 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 the extrusion of noxious molecules, and two "stand-alone" proteins involved in OM biogenesis. This work represents a major advance toward 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.

Keywords: Treponema pallidum; bioinformatics; outer membrane proteins; structural biology; syphilis; vaccines.

FIG 1

The highly flexible POTRA1-5 of T. pallidum BamA lack BamBCDE-interacting residues. (A and B) Ribbon diagrams for the crystal structure of E. coli BamA (PDB accession number 5D0Q) and the structural model of TP0326. Both proteins are in the same orientation. The arrows indicate ECL4 in both proteins. The β-strands (β1 and β16) forming the lateral gate are shown in magenta. The polyserine tract in ECL7 of TP0326 is displayed as green spheres. A nonconservative amino acid substitution (L593Q) in ECL4 of TP0326 is depicted as an orange sphere with the BCE (shown as a blue ribbon). (C and D) The LptD interaction sites (59) in the β-barrel domain of E. coli BamA (PDB accession number 5D0Q) and their equivalent residues in the 3D model of TP0326 are labeled and shown as sticks. (E and F) BamBCDE-interacting residues of the E. coli POTRA arm (PDB accession number 5D0Q) and their equivalent residues in the 3D model of TP0326 POTRA1-5 are depicted as blue, green, cyan, and red surfaces, respectively. Both POTRA arms (E and F) are in the same orientation. (G) R_g distribution of EOM-generated major conformations for the SAXS data of TP0326 POTRA1-5. Three major conformations (compact, intermediate, and extended) for TP0326 POTRA1-5 are shown in the 3D models. (H) The SAXS envelope (gray surface) of TP0326 POTRA1-5 overlaid with the 3D model of its compact conformation (green 3D model in panel G).

FIG 2

Structural model of T. pallidum LptD (TP0515). Shown are cartoon diagrams of the LptD-LptE complex crystal structure (PDB accession number 4Q35) from Shigella flexneri (left) and the 3D structure model of the T. pallidum LptD ortholog (TP0515) (right). Both proteins are in the same orientation. The β-jelly roll N-terminal domain and 26-stranded β-barrel are shown in green and cyan, respectively. LptE of S. flexneri and the C-terminal extension of TP0515 are depicted as orange ribbons. With both proteins, the β-strands (β1 and β26) of the lateral gate and β2 are shown in magenta. Essential amino acids of the lateral gate (N232, P231, and P246) (38) and the intramembrane hole (W180, F203, Y205, V208, F211, and Y212) (75) and their T. pallidum TP0515 equivalents are displayed as sticks. Residues of intramembrane holes are zoomed-in for clarity. The four Cys residues required for the folding of S. flexneri LptD (76) are shown in red. Cys residues in T. pallidum LptD are shown as dark gray sticks.

FIG 3

Eight-stranded β-barrels in T. pallidum. Shown are structural models with BCEs (A), predicted luminal cavities (B), and comparative electrostatics (C) for TP0126, TP0733, TP0479, and TP0698. BCEs are shown as transparent surfaces in panel A. The luminal cavities are shown as gray surfaces in panel B.

FIG 4

Three T. pallidum FadL-like proteins have a bipartite membrane topology. β-Barrel domains are shown in various cyan shades. In all models, the N-terminal hatch regions are shown in magenta. BCEs are depicted as transparent surfaces. Cysteine residues in the cysteine-rich ECL4 of TP0856 and TP0858 are represented as sticks. The C-terminal helical domains of TP0548, TP0859, and TP0865 are shown in orange and indicated by arrowheads. The TPR motifs in the C-terminal domains are shown in black.

FIG 5

trRosetta modeling of full-length Tprs and solution structures of MOSP^N domains of T. denticola MOSP and T. pallidum TprK. (A) MOSP^N and MOSP^C domains of all Tprs, shown in cyan and yellow, respectively. α-Helices of CVRs are red. Residues corresponding to predicted CVR domain boundaries from Pfam are shown as spheres. (B) Ab initio reconstruction of the low-resolution molecular envelopes of T. denticola MOSP and T. pallidum TprK MOSP^N domains (blue and red, respectively) calculated using DAMMIN (166) and DAMAVER (167). Both envelopes were generated without enforcing any symmetry. The maximum dimensions (D_max) and the widths of both envelopes are also labeled.

FIG 6

Channels of T. pallidum OMFs have dissimilar electrostatic potentials. (A) Ribbon diagrams for trimeric structural models of T. pallidum OMFs and the crystal structure of E. coli TolC (PDB accession number 1TQQ). One protomer in all four OMFs is shown in various shades of gold, while the TolC protomer is blue. BCEs are displayed as surfaces. All proteins are in the same orientation, with β-strands labeled. (B) Comparative electrostatics (same orientation) of T. pallidum OMFs and TolC. (C) View from the periplasmic entrance showing the aspartate ring (red spheres) of TP0967 and E. coli TolC. Both proteins are in the same orientation.

FIG 7

Modeling of T. pallidum periplasmic adaptors and TM transporters. (A) Structural models of the T. pallidum periplasmic adaptor (TP0965), the Mac-like TM transporter (TP0962/TP0963/TP964), and the RND-like TM transporter (TP0790) and OMF (TP0969). (B) Cryo-EM structures of E. coli Mac-type (PDB accession number 5NIK) and Acr-type (PDB accession number 5V5S) efflux systems.