Evaluation of the Protective Ability of the Treponema pallidum subsp. pallidum Tp0126 OmpW Homolog in the Rabbit Model of Syphilis

Abstract

In silico analyses of Treponema pallidum subsp. pallidum genomes and predicted proteomes to search for homologs of known bacterial outer membrane proteins (OMPs) led to the identification of tp0126 as a gene encoding a putative member of the OmpW family of porins/virulence factors. Our previous investigations on the role of Tp0126 in T. pallidum biology and syphilis pathogenesis showed that Tp0126 is fully conserved among T. pallidum strains and that transcription of tp0126 is driven by σ⁷⁰ These initial results pointed to a housekeeping function for this protein. We also demonstrated that a guanosine homopolymer of various lengths located between the -10 and -35 consensus sequences in the tp0126 promoter modulates transcription consistently with phase variation, a mechanism that we also previously described for other T. pallidum genes encoding putative OMPs/virulence factors and that is often employed as a strategy for immune evasion. Circular dichroism spectra of recombinant Tp0126 also supported its structural homology with OmpW. Here we further investigated the humoral and cellular responses to Tp0126 during experimental and natural syphilis and the ability of Tp0126 to confer protection against syphilis in immunized rabbits. B-cell epitope mapping showed that compared to sera from experimentally infected animals, immunizations enhanced humoral immunity to sequences located in the putative Tp0126 surface-exposed loops, while phagocytosis assays showed that postimmunization sera opsonized T. pallidum Despite such promising results, no significant protection was seen following infectious challenge in immunized animals versus controls. Functional redundancy and phase variation might explain the lack of effectiveness of this vaccine candidate and/or design.

Keywords: OmpW; Tp0126; Treponema pallidum; functional redundancy; phase variation.

FIG 1

(A) Titration of prechallenge sera from each rabbit. (B) B-cell epitope mapping performed with postimmunization sera. Bars relative to peptides containing putative surface-exposed amino acids are in gray, while bars relative to peptides that map onto the predicted Tp0126 β-scaffolding are in white. Gray/white bars contain amino acids that map to both the predicted surface-exposed loops and the scaffolding. FL, full-length peptide. (C) Tp0126 sequences recognized by prechallenge rabbit immune sera, highlighted in red in the Tp0126 structural model. (D) Opsonophagocytosis assay performed using pooled prechallenge sera from Tp0126-immunized rabbits. IRS, day 90 immune serum from rabbits infected i.t. with the T. pallidum Nichols strain; NRS, noninfected rabbit serum.

FIG 2

(A) Measurement of indurated lesion diameters at challenge sites. (B) Percentage of ulcerating lesions. Open circles represent Tp0126-immunized rabbits; solid circles are unimmunized control rabbits. (C) Dark-field microscopy counts of treponemal cells in aspirates from challenge lesions. Needle aspirates were obtained from each lesion at day 13 postchallenge. (D) Molecular evaluation of treponemal burden within challenge lesions. Burden was measured by quantification of T. pallidum tp0574 mRNA normalized to the rabbit HPRT message.

FIG 3

B-cell epitope mapping using synthetic peptides. (A to C) Peptides (20-mers overlapping by 10 aa) based on the Tp0126 sequence (Table 1) were used along with sera collected at day 30 (A), day 60 (B), and day 90 (C) from rabbits infected i.t. with the Nichols strain of T. pallidum. Bars represent the mean OD readings after background (from no-antigen wells) subtraction. (D) Reactivity of sera (n = 7) from latent syphilis patients. Bars relative to peptides that contain putative surface-exposed amino acids are in gray, while bars relative to peptides that map onto the predicted Tp0126 β-scaffolding are in white. Gray/white bars contain amino acids that map to both the predicted surface-exposed loops and the scaffolding. (E) Tp0126 sequences recognized by day 90 immune rabbit sera, highlighted in yellow in the Tp0126 three-dimensional (3D) model. (F) Tp0126 sequences recognized by human sera. Sequences are highlighted in orange in the Tp0126 3D model. The Tp0126 3D model was generated using I-TASSER as described previously (24). FL, full-length peptide.

FIG 4

(A) T-cell proliferation assay with splenic lymphocytes. Splenocytes were harvested from rabbits at day 90 after experimental i.t. infection using test antigens (rTp126 and Tp0126-derived synthetic peptides), the control antigen concanavalin A (ConA), and no antigen (No Ag). Bars represent the geometric means ± standard errors of means of quadruplicate experimental values for each antigen for three rabbits. Significant differences (P ≤ 0.05) compared to the no-antigen value are indicated by an asterisk. (B) Tp0126 sequences that induced proliferation. Sequences are highlighted in green in the Tp0126 3D model. The Tp0126 3D model was generated using I-TASSER as described previously (24).

FIG 5

(A) Distribution of poly(G) lengths. Distributions were determined in the inoculum treponemes as well as in treponemes in lesions from Tp0126-immunized and control rabbits. G length percentages are 61.2% (9 G’s), 37.1% (10 G’s), and 1.7% (11 G’s) in the challenge inoculum; 36.8% (9 G’s), 58.2% (10 G’s), and 5.0% (11 G’s) in Tp0126-immunized rabbits; and 54.6% (9 G’s) and 45.4% (10 G’s) in control samples. (B) tp0126 mRNA levels. Message levels are normalized to the tp0574 message in the T. pallidum challenge inoculum and in treponemes from postchallenge lesions of unimmunized and Tp0126-immunized rabbits.