Fig 1.
SAXS analysis of full-length TP0751.
(A) Ribbon diagram of the bipartite structural model of full-length TP0751 generated using the intensive mode of Phyre2 [56]. The unstructured N-terminal region (residues 25–96) and C-terminal lipocalin domain (residues 97–237) are shown in pink and green, respectively. (B) Plots showing the log of the scattering intensity (I) as a function of momentum transfer (q = 4πsin(θ)/λ). The black circles are SAXS experimental data; the colored curves are theoretical scattering profiles calculated from different structural models using FoXS [123]. The χ2 shown in parentheses indicate the fit of the theoretical scatterings to the experimental SAXS data. (C) Comparison of the normalized inter-atomic pairwise distribution function (P(r)), computed from the experimental SAXS data (black circles) and different 3D models (colored lines). The P(r) functions show that TP075125-237 has a bipartite architecture with a diameter of approximately 75Å.
Fig 2.
The N-terminal region of TP0751 is intrinsically disordered.
(A) Prediction of disorder propensity along the length of TP075125-237 using IUPred2 [58], PrDOS [57], PONDR [131]. The dashed line represents the domain boundary of TP075125-237. (B) Ribbon diagrams show ten NMA- generated conformations of the bipartite model of TP075125-237. The best optimized is shown in yellow and indicated by the arrow. (C) Ab initio reconstruction of the molecular envelope (gray surface) of TP075125-237 calculated from the SAXS data and overlaid on the NMA optimized structural model.
Table 1.
Calycin/lipocalin structural orthologs of TP0751.
Fig 3.
Structural mapping of the predicted ligand-binding regions of TP075197-228.
(A) The left panel shows selected ligands and their docking energies. Dashed boxes indicate the predicted binding regions; residues predicted to interact with individual ligands are underlined. (B) Close-up of the predicted ligand-binding sites. The residues predicted to form the four binding regions are depicted as sticks and colored as in panel A. Molecular docking snapshots of predictions for binding of (C) heme and (D) cholesterol.
Fig 4.
The lipocalin domain of TP0751 binds heme.
UV/Vis differential spectra of (A) TP075197-237 (5 μM), (B) TP075125-96 (5 μM), and (C) lysozyme (10 μM) incubated with graded concentrations of heme. The arrow in panel A denotes absorption maxima; the dashed lines at 400 and 500 nm in all three panels denote the spectral range for band broadening [132]. The changes in absorbance of the Soret peak (416 nm) plotted against heme concentrations (0–30 μM) were used to determine the dissociation constant (Kd, 11.7 ± 2.2 μM) for TP075197-237, using a one-site binding model with Hill slope (Inset, panel A).
Fig 5.
TP075197-228 and Tpp17 share similar structural topologies.
The upper panel shows ribbon diagrams for the structures of TP075197-228 (PDB ID: 5JK2), Tpp17 (PDB ID:4U3Q), and the N-terminal domain (residues 21–103) of E. coli NlpE (PDB ID:4Z4H) (all in the same orientation). The Ω-type loops are shown in orange. Residues of the calycin signature motif are shown as blue sticks and labeled. The lower panel presents secondary structure topology diagrams. β-strands and α-helices are represented by arrows and cylinders, respectively. N- and C-termini are indicated, and the ‘Ω-type’ loops shared by TP075197-228 and Tpp17 are labeled.
Fig 6.
Native TP0751 is a low abundance lipoprotein in T. pallidum.
(A) Schematic depiction of the tp0750-tp0751 operon. Primers used for PCR amplification of tp0750, tp0751 and the intergenic region (S2 Table) are indicated by purple, blue and pink arrows, respectively. (B) Transcript copy numbers normalized to flaA. Error bars indicate standard errors of the mean (3 biological replicates, assayed in quadruplicate). p values for pairwise comparisons were determined using a two-tailed t test. (C) Trinity Biotech MARBLOT T. pallidum lysate strips were incubated overnight at 4° C with polyclonal rat antisera (1:1,000) against TP0750, TP075125-237, Tpp17, p30.5 (TP0453), and Tpp47 or normal rat serum (NRS) followed by goat anti-rat IgG HRP conjugate (1:30,000) for 1 h at RT. Strips were aligned and developed on a single sheet of film using the SuperSignal West Pico chemiluminescent substrate. Arrows indicate the presumptive monomers of TP0750 and TP0751, the known monomer of Tpp17, p30.5, and Tpp47. The asterisk indicates Tpp17 dimers [140]. The degradation products of Tpp47 have been described previously [87, 141].
Fig 7.
TP0751 weakly induces antibodies during experimental and human syphilis.
Immunoblot reactivities of sera from (A) rabbits immune to re-infection with the Nichols, Mexico A or SS14 strains and (B) five HIV-negative patients with secondary syphilis (SS) against Tpp17, Tpp47 and TP075125-237 (100 ng of each protein, 1° Ab 1:250). Molecular mass standards (in kilodaltons) are indicated on the left.
Fig 8.
Immunization with TP075125-237 does not attenuate lesion development or prevent dissemination of spirochetes.
(A) Cutaneous lesions for TP075125-237- and sham-immunized rabbits at sacrifice, 23 days post-challenge. (B) Average lesion circumference (mm) and (C) percentage of lesions ulcerating. No statistically significant differences were found in lesion circumference (nonlinear regression and extra sum of squares F-test) or lesion ulceration (multiple row t tests) between the two groups. Spirochete burdens in (D) cutaneous lesions, (E) spleens and (F) livers at the time of sacrifice (23 days post-challenge) assessed by qPCR (polA transcripts normalized to β-actin). Bars indicate the means of four TP0751-immunized and two sham-immunized animals. T. pallidum burdens in a punch biopsy of each cutaneious lesion and triplicate samples of liver and spleen from each rabbit were assayed by qPCR. p values for pairwise comparisons were determined using a two-tailed t test. (N.S. = not significant).