Antigenic and structural characterization of Treponema pallidum (Nichols strain) endoflagella.

Purified endoflagella from Treponema pallidum, Nichols strain, were characterized both structurally and antigenically. Structural analysis showed T. pallidum endoflagella are composed of 35- and 33-kilodalton (kDa) subunits which lack cysteine and do not share N-terminal amino acid sequence homology (20 residues). Intact endoflagella were dissociated into the composite subunits by incubation, which disrupts noncovalent bonds. Antiserum raised against purified T. pallidum endoflagella identified shared epitopes on the endoflagellar polypeptides of the nonpathogen, Treponema phagedenis biotype Reiter. Pathogen-specific epitopes were also found on the 35- and 33-kDa polypeptides by using affinity-purified endoflagellar antibodies. The pathogen-specific epitopes were localized by immunoblotting analysis of chymotryptic digests of the endoflagellar subunits; 18- and 26-kDa fragments derived from the 35-kDa subunit were found to possess a majority of the pathogen-specific epitopes. Both the 35- and 33-kDa subunits had surface exposure, as determined by immunoelectron microscopy, although additional immunochemical data indicated that the surface exposure of the 35-kDa subunit was greater.     

Extensive cross reactivity between Treponema pallidum and cultivable treponemes demonstrated by sequential immunoadsorption

Extensive cross reactivity between Treponema pallidum and three nonpathogenic species of treponemes was demonstrated by the Western blot technique. Rabbit antiserum produced by adjuvant immunization with solubilized T. pallidum antigens reacted with 34 T. pallidum antigens and with approximately 30 antigens each of T. phagedenis biotype Reiter, T. noguchii and T. vincentii. Adsorption of the antiserum with T. phagedenis Reiter removed only about half of the cross-reacting antibodies. Sequential adsorption with all three nonpathogenic treponemes removed antibodies to all but three polypeptides of 36,000, 34,000 and 27,000 daltons.     

Opsonization of Treponema pallidum is mediated by immunoglobulin G antibodies induced only by pathogenic treponemes.

Rabbit antisera to Leptospira interrogans, Borrelia hermsii, and Treponema phagedenis biotype Reiter, reactive to shared spirochetal antigens, failed to enhance phagocytosis of Treponema pallidum by macrophages, while immunoglobulin G to Treponema pallidum subsp. pertenue and Treponema paraluiscuniculi promoted phagocytosis. Opsonic antibodies are directed to pathogen-restricted, not shared spirochetal, antigens.     

Development of monoclonal antibodies that recognize Treponema pallidum.

We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay.     

Molecular characterization of common treponemal antigens.

A molecular characterization of cross-reactive antigens of Treponema pallidum Nichols and Treponema phagedenis biotype Reiter that are reactive with normal and syphilitic human sera is described. At least 8 common polypeptides, 14 T. pallidum-specific antigens, and 2 T. phagedenis biotype Reiter-specific antigens were identified.     

Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum.

Pathogen-specific membrane immunogens of Treponema pallidum subsp. pallidum (T. pallidum) have been identified previously by phase partitioning with the nonionic detergent Triton X-114. One of these antigens, a 15-kilodalton (kDa) polypeptide, is expressed in relatively small quantities in T. pallidum but is highly immunogenic in both human and experimental syphilis. The native T. pallidum antigen was purified to homogeneity from the mixture of Triton X-114 detergent-phase proteins by chromatofocusing. Recombinant Escherichia coli clones were selected from a T. pallidum genomic DNA library by using monoclonal antibodies specific to the 15-kDa antigen; immunoblotting and minicell analyses confirmed expression of the 15-kDa protein in the transformants. Southern hybridization with a 1.1-kilobase fragment of DNA encoding the 15-kDa-antigen gene indicated that the gene is probably present in a single copy within the genomes of both T. pallidum and T. pallidum subsp. pertenue (the agent of yaws), while it is absent from the genome of the nonpathogenic Treponema phagedenis biotype Reiter. Cell fractionation studies with Triton X-114 demonstrated that the recombinant polypeptide possesses hydrophobic properties similar to those of the native antigen and localized the cloned 15-kDa antigen to the inner membrane of E. coli. Protein processing experiments in minicells revealed that a precursor appears to be processed to the mature 15-kDa polypeptide.     

Molecular specificities of monoclonal antibodies directed against virulent Treponema pallidum.

Radioimmunoprecipitation (RIP) and Western blot analyses with specific anti-Treponema pallidum subsp. pallidum monoclonal antibodies were used to identify antigens with apparent masses of 102, 84, 54, 53, 52, 47, 32, 29, and 24 kilodaltons (kDa). Cross-reactivity of these antibodies with T. pallidum subsp. pertenue antigens and lack of cross-reactivity with T. phagedenis biotype Reiter, T. vincentii, T. refringens, T. scoliodontum, and T. denticola were also demonstrated by RIP and Western blot analyses. Reactivities in the T. pallidum immobilization test, along with the RIP of lactoperoxidase-catalyzed iodination products, suggested that the identified antigens were surface associated. The abundance and surface association of the 47- and 84-kDa antigens were supported by reactivity in the microhemagglutination test for T. pallidum and by strong reactivity of monoclonal antibodies upon indirect immunofluorescence assays with rabbit-cultivated T. pallidum subsp. pallidum, respectively, but not with T. phagedenis biotype Reiter. Anti-47-kDa and anti-84-kDa monoclonal antibodies were also reactive in indirect immunofluorescence assays using treponemes found in dark-field-positive smears of human genital ulcers.     

Identification and localization of integral membrane proteins of virulent Treponema pallidum subsp. pallidum by phase partitioning with the nonionic detergent triton X-114.

Integral membrane proteins of Treponema pallidum subsp. pallidum (T. pallidum) were identified by phase partitioning with the nonionic detergent Triton X-114; antigens with apparent molecular masses of 47, 38, 36, 34, 32, 17, and 15 kilodaltons (kDa) were identified in the detergent phase. Immunoblotting with murine monoclonal antibodies directed against pathogen-specific 47- and 34-kDa T. pallidum antigens confirmed their presence in the detergent phase. Endoflagellar proteins of T. pallidum were not detected in immunoblots of detergent-phase proteins when monospecific antisera directed against endoflagella of the nonpathogenic T. phagedenis biotype Reiter were used. At detergent concentrations (0.02 and 0.1%) which appeared to solubilize selectively the outer membranes of treponemes radiolabeled with 35S in vitro, limited amounts of detergent-phase proteins were immunoprecipitated. Greater amounts of detergent-phase proteins were extracted at higher detergent concentrations (0.5 and 2.0%) which resulted in both outer membrane solubilization and ultrastructural derangements of the residual cytoplasmic bodies. Furthermore, Triton X-114 extraction of both intact treponemes and organisms without outer membranes yielded detergent phases with similar protein profiles. The results of these experiments indicate that the hydrophobic proteins identified by Triton X-114 are not located exclusively in the T. pallidum outer membrane. The results are also consistent with the hypothesis that the T. pallidum outer membrane is a protein-deficient lipid bilayer.     

Immunogenicity of three recombinant Treponema pallidum antigens examined in guinea pigs

The immunogenicity of recombinant treponemal antigens TmpA, TmpB and TmpC incorporated in RIBI adjuvant and injected into inbred strain 2 guinea pigs has been examined. The immune status of these animals has been challenged by infection with Treponema pallidum, Nichols. The immune response evaluated by the fluorescent-antibody test, microhemagglutination test and ELISA demonstrated high titers of antibodies to the T. pallidum antigens. The immunoblot analysis proved that the antibodies were directed to the 43-(Tmp A) 34- (Tmp B) and 35-kdalton (Tmp C) polypeptides. Antibodies cross-reacting with Treponema phagedenis biotype Reiter were, however, also detected. In spite of high titers of antibodies the animals were not protected against challenging infection with 10(8) organisms of T. pallidum.     

Lymphocyte transformation in inbred guinea pigs infected with Treponema pallidum nichols

T lymphocytes were purified from peritoneal exudates from young male, inbred strain 2 guinea pigs infected with 8 X 10(7) Treponema pallidum Nichols and from control animals injected with normal rabbit testes extract. Groups of animals were sacrificed after 15, 30, 60, and 90 days of infection, and the cells were analyzed for their proliferative response to concanavalin A and phytohemagglutinin and to sonicated T. pallidum, Treponema phagedenis biotype Reiter, and normal rabbit testes extract. No significant differences were observed in the responses to mitogens. Cells from infected but not from control animals responded with significant proliferation to T. pallidum antigen, but neither infected nor control cells reacted to T. phagedenis Reiter antigen. Although cells from both infected and control animals responded to normal rabbit testes extract, this response in the infected animals was transient and much lower than the response to T. pallidum antigen.     

Genetics of Treponema: characterization of Treponema hyodysenteriae and its relationship to Treponema pallidum.

Saturation reassociation assays with 125I-labeled treponemal DNAs show that Treponema hyodysenteriae is genetically unrelated to T. pallidum (Nichols), T. phagedenis biotype Reiter, and T. refringens biotype Noguchi. Pathogenic and nonpathogenic isolates of T. hyodysenteriae exhibited 28% sequence homology and had an extremely low guanine-plus-cytosine content (25.8%).     

Pathogen specificity of Treponema pallidum subsp. pallidum integral membrane proteins identified by phase partitioning with Triton X-114.

The antigenically conserved proteins of Treponema pallidum subsp. pallidum and four nonpathogenic cultivatable treponemes were investigated by phase partitioning with the nonionic detergent Triton X-114 and immunoblot analysis. None of the T. pallidum integral membrane proteins identified by phase partitioning (detergent-phase proteins) appeared to be antigenically related to proteins of the nonpathogens. Protease-resistant material similar to lipopolysaccharide was identified in the detergent phase from T. phagedenis biotype Reiter but was not detected in T. pallidum.     

Humoral response in Treponema pallidum-infected guinea pigs: I. Antibody specificity.

Young male inbred strain 2 guinea pigs were infected intradermally with 8 X 10(7) Treponema pallidum extracted from a rabbit orchitis, and 5 months later reinfected with 10(7) T. pallidum. Ninety percent of the animals developed symptomatic lesions after initial infection but none on challenge. Immunoblotting of sera obtained at intervals after infection or reinfection showed antibodies against T. pallidum antigen (TP), nonpathogenic treponemes--T. phagedenis biotype Reiter (TR), T. refringens strain Noguchi (TN), and T. vincentii (TV)--as well as normal rabbit serum (NRS) and normal rabbit testes extract (NRT). Antibodies reacting with TP were detected as early as 17 days (five polypeptides) and steadily rose (at 3 months 17 polypeptides were seen). Cross-reacting antibodies to TR, TN, TV, or rabbit proteins decreased within 3 to 5 months. After reinfection, the antibodies to NRS increased more sharply than the anti-treponemal antibodies. Adsorption with TR and NRS of sera obtained after infection or reinfection produced a reduction of antibodies to TP by 75-87%.     

Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins.

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

Molecular characterization of glycoprotein antigens on surface of Treponema pallidum: comparison with nonpathogenic Treponema phagedenis biotype Reiter.

Four glycoproteins of Treponema pallidum were identified by intrinsic [14C]glucosamine labeling. Only two glycoproteins were demonstrated in T. phagedenis biotype Reiter with the same technique. Glycoproteins of both treponemes were characterized as antigens and shown to be localized within the outer membranes of the microorganisms.     

Capacity of virulent Treponema pallidum (Nichols) for deoxyribonucleic acid synthesis.

Treponema pallidum (Nichols) was extracted from infected rabbit tissue, and cell lysates were prepared for monitoring thymidine kinase and deoxyribonucleic acid polymerase activities. No thymidine kinase could be demonstrated in preparations of T. pallidum or the cultivable T. phagedenis biotype Reiter. Significant levels of deoxyribonucleic acid polymerase were detected in both treponemal samples. Interestingly, comparisons of polymerase activity among a spectrum of bacterial genera revealed a direct correlation between enzyme concentrations and estimated generation time. Incorporation of [3H]uridine and [3H]thymidine into macromolecules by intact T. pallidum and the Reiter treponeme was examined. Selective ribonuclease-deoxyribonuclease digestion and cesium chloride gradient banding demonstrated that T. pallidum, independent of the host, and T. phagedenis were capable of synthesizing deoxyribonucleic acid only from the [3H]-uridine precursor.     

Changes in the cell surface properties of Treponema pallidum that occur during in vitro incubation of freshly extracted organisms

We previously reported that a number of Treponema pallidum membrane proteins appear to reside on the cell surface, since intact treponemes radiolabeled by overnight incubation in medium containing [35S]methionine bind immunoglobulin G (IgG) antibodies directed against these proteins. In the present study, it was found that freshly extracted organisms radiolabeled in vitro for only 2 h inefficiently bound IgG antibodies directed against just two proteins of molecular weights 40,000 and 34,000. An in vitro incubation period of greater than 8 h was required before IgG antibodies present in rabbit syphilitic serum could recognize additional protein antigens on the cell surface. Treatment of aged treponemes, but not freshly extracted organisms, with 0.04% sodium dodecyl sulfate selectively removed a membranous layer from the treponemal surface. Only three treponemal proteins were found associated with this structure, including the same 40,000- and 34,000-molecular-weight proteins mentioned above. These two proteins most likely represent endoflagellar subunits, since they were precipitated with rabbit antisera prepared against purified endoflagellar subunits of the cultivable treponemal strain Treponema phagedenis. Further evidence also was obtained that cells of T. pallidum actively secrete into their extracellular environment a unique class of low-molecular-weight proteins.     

Immunodiagnostic test for detection of serum antibody to Treponema pallidum (syphilis): fibronectin as a capture vehicle for treponemal adhesins

Knowledge that Treponema pallidum adhesin proteins bind to host fibronectin (Fn) via ligand-receptor interactions has resulted in development of an ELISA for measuring specific antitreponemal antibodies in sera of syphilitic patients and infected experimental animals. As little as 50 ng of T. pallidum total protein extract added to Fn-coated wells permitted half-maximal levels of ELISA reactivity. Detection of serum antibody from intratesticularly infected rabbits occurred at dilutions greater than 1/100,000. Antibody titers in serum from patients with primary and latent syphilis were positive at 1/1 000 dilutions while serum samples from patients with secondary syphilis were reactive at 1/10,000. Furthermore, the ELISA proved useful for evaluating serum samples from individuals with other treponemal infections. Antibodies raised against the non-pathogenic spirochete, T. phagedenis biotype Reiter, were non-reactive with Fn-T. pallidum complexes. Also, Reiter treponemal proteins did not bind to Fn-coated wells. This ELISA using Fn as a capture vehicle for treponemal adhesin proteins was superior to 3 other routinely used tests for syphilis diagnosis.     

Experimental congenital syphilis: guinea pig model.

Neonates born to female guinea pigs of either a highly susceptible (C4D) or a resistant (Albany) strain, infected prior to or during pregnancy with a single dose of Treponema pallidum, showed in their sera from the first day of life immunoglobulin M (IgM) antibodies to T. pallidum, circulating immune complexes consisting of IgM antibodies and treponemal antigens, and IgM rheumatoid factor. Although the animals were asymptomatic for a 6-month observation period, several lines of evidence indicated that they were infected in utero. Molecular analysis of whole sera, purified serum IgM fraction, or dissociated immune complexes demonstrated IgM reactivity against one (47 kDa) or more of several T. pallidum peptides (15, 17, 37, 42, 45, and 87 kDa) recognized as integral membrane components. Sequential analysis of the neonates' sera by immunoblot and enzyme-linked immunosorbent assay, using alcohol-treated T. pallidum, T. phagedenis biotype Reiter, and T. vincentii, demonstrated early IgM antibodies followed 3 to 4 months later by IgG2- and IgG1-specific antibodies to T. pallidum. Moreover, an infectivity test done in five rabbits with pooled tissue extracts prepared from liveborn or stillborn animals evoked a seroconversion in two rabbits (reactive Venereal Disease Research Laboratory and fluorescent treponemal antibody tests), suggesting the presence of T. pallidum in the organs. Sera from neonates born to either T. phagedenis biotype Reiter-injected mothers or three normal pregnant females were all serologically negative. The model offers new possibilities for exploration of factors responsible for asymptomatic infection often observed in human congenital syphilis.