Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response

Abstract

We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

Figure 1

Alignments of predicted amino acid sequences of tpr genes of subfamilies I, II, and III using the Clustal W program (41). Dark shaded areas indicate sequence conservation among the Tpr proteins. (A) Alignment of subfamily I (Tpr C, D, F, and I). (B) Alignment of subfamily II (Tpr E, G, and J). (C) Alignment of subfamily III (Tpr A1, A2, B, H, K, and L). The sequences of the genome-derived tpr genes can be found in Genbank (under Accession No. AE 000520) or in the T. pallidum web site at http://utmmg.med.uth.tmc.edu/treponema/tpall.html

Figure 1

Alignments of predicted amino acid sequences of tpr genes of subfamilies I, II, and III using the Clustal W program (41). Dark shaded areas indicate sequence conservation among the Tpr proteins. (A) Alignment of subfamily I (Tpr C, D, F, and I). (B) Alignment of subfamily II (Tpr E, G, and J). (C) Alignment of subfamily III (Tpr A1, A2, B, H, K, and L). The sequences of the genome-derived tpr genes can be found in Genbank (under Accession No. AE 000520) or in the T. pallidum web site at http://utmmg.med.uth.tmc.edu/treponema/tpall.html

Figure 1

Alignments of predicted amino acid sequences of tpr genes of subfamilies I, II, and III using the Clustal W program (41). Dark shaded areas indicate sequence conservation among the Tpr proteins. (A) Alignment of subfamily I (Tpr C, D, F, and I). (B) Alignment of subfamily II (Tpr E, G, and J). (C) Alignment of subfamily III (Tpr A1, A2, B, H, K, and L). The sequences of the genome-derived tpr genes can be found in Genbank (under Accession No. AE 000520) or in the T. pallidum web site at http://utmmg.med.uth.tmc.edu/treponema/tpall.html

Figure 2

Hydropathy plot for Tpr K determined using the TMpred program (http://ulrec3.unil.ch/software/TMPRED). The x-axis represents the amino acid residues in the sequence, and the y-axis indicates the hydropathy index. Negative values indicate hydrophilic characteristics, and positive values indicate hydrophobic characteristics. This analysis shows two putative transmembrane hydrophobic regions with a large intervening hydrophilic domain.

Figure 3

Ethidium bromide– stained gel showing PCR amplification of Nichols strain cDNA using primers for unique sequences of the variable domains of tpr A–L. The lane labeled “+” shows the result of amplification using control primers (demonstrates sufficient amounts of target cDNA in the sample) and lanes 1–12 show the results when amplification was performed using primers specific for each tpr gene. Total RNA was extracted from a known number of freshly harvested Nichols strain treponemes, followed by treatment of the RNA sample with RNAse free DNAse A. First-strand cDNA was made by random priming, and PCR amplification was performed using primers specific for each tpr gene (Table I). DNAse A treatment of the RNA samples, lack of amplification of RNA without cDNA synthesis, and determination of primer efficiency ruled out the possibility that these results represent either DNA contamination or PCR efficiency artifacts (data not shown).

Figure 4

Opsonization of T. pallidum by antisera to recombinant Tpr K variable domain. Columns represent the percentage of rabbit peritoneal macrophages ingesting T. pallidum, Nichols strain, after 4 h of incubation with viable T. pallidum (107 treponemes and 2 × 10⁶ macrophages) in RPMI with 10% final concentration of NRS plus 1% final concentration of test or control antiserum. Ingested treponemes were visualized by indirect immunofluorescence staining. Triplicate cultures were prepared for each experiment and were scored for each condition by a blinded observer. Column values represent means ± SEM of four separate experiments. Significant opsonization is determined by comparison with the NRS values (Student's t test) and P values are shown. IRS, pooled syphilitic immune rabbit sera; Tpr K, anti-recombinant Tpr K variable domain.

Figure 5

Intradermal challenge in a rabbit immunized with the recombinant Tpr K variable domain (A) and a normal unimmunized (B) rabbit. The immunized rabbit was injected with 125 μg purified recombinant peptide in Ribi adjuvant (MPL + TDM + CWS), divided between intramuscular, intradermal, subcutaneous, and intraperitoneal sites, and similarly boosted at 3 and 6 wk. The rabbits were challenged intradermally with 10⁵ T. pallidum Nichols strain at each of eight sites 3 wk after the final immunization. Black ink marks are seen to the left of the challenge sites.