Biomedical subjects

J D Radolf

Publications and source records attributed to J D Radolf.

At least 109 records · Page 6Linked to original sources

PubMed1989

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.

Antibodies, Monoclonal↗

PubMed1989

Penicillin-binding proteins and peptidoglycan of Treponema pallidum subsp. pallidum.

Penicillin-binding proteins (PBPs) of Treponema pallidum subsp. pallidum (T. pallidum) were characterized by using [3H]penicillin G and a conjugate consisting of ampicillin and 125I-labeled Bolton-Hunter reagent. Both antibiotics specifically radiolabeled proteins with molecular masses of 94, 80, 63, and 58 kilodaltons (kDa); 125I-labeled Bolton-Hunter reagent-ampicillin also radiolabeled several polypeptides with lower molecular masses. The 94- and 58-kDa proteins demonstrated the highest binding affinities for [3H]penicillin G and were radiolabeled at concentrations of 8 and 40 nM, respectively. Radiolabeling of PBPs was detectable after 1 min of incubation in 1 microM [3H]penicillin G and was nearly maximal within 10 min. The rapidity of penicillin binding contrasted with the observation that only 40% of virulent treponemes became immobilized during prolonged incubation in vitro with a much higher concentration (1 mM) of unlabeled penicillin. Two lines of evidence indicated that most, if not all, of the PBPs are integral cytoplasmic membrane proteins: (i) preincubation of organisms in 0.1% Triton X-100 solubilized nearly all of the outer membranes but did not affect radiolabeling of PBPs, and (ii) except for the 80-kDa protein, the PBPs partitioned into the detergent phase following extraction with the nonionic detergent Triton X-114. The presence of peptidoglycan in T. pallidum was confirmed by the detection of muramic acid in the sodium dodecyl sulfate-insoluble, proteinase K-resistant residue obtained from Triton X-114-extracted organisms.

Animals↗

PubMed1989

Antigenic evidence for simultaneous expression of two different lipooligosaccharides by some strains of Haemophilus influenzae type b.

Isolates of Haemophilus influenzae type b (Hib) can be divided into three antigenic groups based on their reactivities with a set of two monoclonal antibodies (MAbs) directed against epitopes in the oligosaccharide region of Hib lipooligosaccharide (LOS) (P. A. Gulig, C. C. Patrick, L. Hermanstorfer, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 55:513-520, 1987). Approximately 24% of Hib strains react with both of these LOS-specific MAbs. Immunoprecipitation experiments involving several of these strains indicated that the epitopes recognized by these MAbs resided in two different LOS molecules, both of which were synthesized by these particular Hib strains. In addition, Western blot (immunoblot) analysis of proteinase K-treated cell extracts of these strains that had been subjected to sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis revealed two different LOS staining patterns when they were probed independently with the two MAbs. Colony blot radioimmunoassay of hundreds of colonies of one of these Hib strains showed that each colony bound both MAbs. Immune electron microscopy confirmed that individual cells of this same Hib strain expressed both types of LOS molecule at the same time. An antibody accessibility radioimmunoassay was used to show that different Hib strains of this type varied in the relative amounts of each of the two MAbs that they could bind to their cell surfaces. These findings indicate that some Hib strains can synthesize two antigenically distinct LOS molecules simultaneously.

Antibodies, Bacterial↗

PubMed1989

Major integral membrane protein immunogens of Treponema pallidum are proteolipids.

A number of the major pathogen-specific immunogens of Treponema pallidum were characterized recently as amphiphilic, integral membrane proteins by phase partitioning with Triton X-114 (J. D. Radolf, N. R. Chamberlain, A. Clausell, and M. V. Norgard. Infect. Immun. 56:490-498, 1988). In the present study, we demonstrated that the same membrane immunogens (designated as detergent phase proteins [DPPs]) become radiolabeled upon in vitro incubation of T. pallidum with various 3H-labeled fatty acids. Radioimmunoprecipitation with a monoclonal antibody confirmed that the 3H-labeled 47-kilodalton protein corresponded to the well-characterized treponemal antigen with the identical apparent molecular mass. Failure to detect 3H-labeled DPPs following incubation with erythromycin confirmed that protein acylation required de novo protein synthesis by the bacteria. When treponemes were incubated with [3H]myristate, [3H]palmitate, or [3H]oleate, radiolabeled proteins corresponding to the DPPs were detected upon autoradiography. Demonstration that a number of the abundant membrane immunogens of T. pallidum are proteolipids provides information to help clarify their membrane association(s) and may serve to explain their extraordinary immunogenicity.

Animals↗

PubMed1989

Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity.

The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid. Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T. pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid. Both intact E. coli and E. coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E. coli corresponds to the cloned T. pallidum antigen. Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems. The products synthesized by T. pallidum, intact E. coli, or E. coli minicells were hydrophobic, while the protein synthesized in an E. coli cell-free translation system was hydrophilic. Processing experiments with E. coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids. These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s). The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T. pallidum.

Acylation↗

PubMed1989

Effect of primary immunization on pulmonary clearance of nontypable Haemophilus influenzae.

Nontypable Haemophilus influenzae (NTHI) is being increasingly recognized as a cause of both adult pneumonia and acute infectious exacerbations in chronic bronchitis. We used a mouse model to study the immune enhancement of pulmonary clearance of NTHI after a primary immunization. BALB/c mice were immunized with whole NTHI either by intraperitoneal (i.p.) or intratracheal (i.t.) routes. There was 10-fold more NTHI-directed antibody detected in the serum of the i.p.-immunized mice than in the serum from the i.t.-immunized animals. Western blot analysis revealed that these antibodies were directed against both NTHI lipooligosaccharide and the various outer membrane proteins of NTHI. The development of NTHI-directed antibodies in serum was associated with significant enhancement of early pulmonary clearance of NTHI. Six hours after delivery of an endobronchial challenge with NTHI, the i.p.-immunized mice had cleared most of the organisms from their lungs, while the i.t.-immunized mice did not clear NTHI any more rapidly than did unimmunized mice. Serum from the i.p.-immunized mice caused more than 99% uptake of NTHI in an in vitro opsonophagocytic assay, while serum from i.t.-immunized mice stimulated little or no phagocytosis of this organism. Opsonophagocytosis of NTHI was obtained with bronchoalveolar lavage (BAL) fluid collected from i.p.-immunized mice 6 h after, but not before, an endobronchial challenge with NTHI. Intravenous injection of an opsonic IgG monoclonal antibody directed against NTHI lipooligosaccharide resulted in both the appearance of this antibody in the alveolar spaces of the unperturbed lung and enhanced pulmonary clearance of NTHI. These data indicate that the i.p. (systemic) route of immunization is more effective than the i.t. route in establishing pulmonary immunity to NTHI in this model system. Furthermore, immune enhancement of clearance of NTHI from the lungs after a primary immunization apparently results from the exudation of opsonic and bactericidal antibodies from the serum into the alveolae in response to the inflammatory challenge.

Animals↗

PubMed1988

Immunization of rabbits with recombinant Treponema pallidum surface antigen 4D alters the course of experimental syphilis.

Rabbits immunized with the protease resistant recombinant Treponema pallidum surface-associated Ag 4D showed an altered course of experimental syphilis after intradermal challenge with virulent T. pallidum. Vaccination trials using three different protocols were examined. In one experiment, a combined i.m.-i.v. immunization protocol was compared with an i.m. injection schedule. Four of five rabbits immunized by the i.m.-i.v. protocol developed morphologically atypical lesions at each of 16 sites, whereas the i.m. injected animals showed no evidence of attenuated disease. Moreover, immunization by both protocols elicited equally high ELISA titers of 4D-specific IgG antibody suggesting that cellular immune mechanisms may have been involved. In an effort to augment cell-mediated responses to 4D, seven rabbits received i.v. immunizations with an emulsion of 4D, purified BCG cell wall skeletons, and trehalose dimycolate in an oil-microdroplet form (4D-cell wall skeleton-trehalose dimycolate). Splenic lymphocytes from three representative immunized animals had a strong in vitro proliferative response to 4D as compared to controls. Further, all animals developed high anti-4D titers in response to immunization. After challenge with virulent T. pallidum, six of the seven rabbits developed an early cutaneous response (3-8 days) at the sites of inoculation consistent with a hypersensitivity reaction followed by morphologically atypical lesions. Aspirates from each of three representative atypical lesions were devoid of treponemes by darkfield examination at day 14, whereas motile T. pallidum were observed in aspirates from four of five control sites. Between days 20 and 24, lesions in the 4D-cell wall skeleton-trehalose dimycolate immunized rabbits began to enlarge and become more typical in appearance; aspirates from six of seven representative lesions were darkfield positive. We conclude that 1) immunization with the r4D Ag alters the course of experimental syphilis in a manner consistent with previously defined parameters of partial protection and 2) immunizations protocols containing an i.v. component are more effective than protocols using the i.m. route alone.

Animals↗

PubMed1988

Unusual manifestations of secondary syphilis and abnormal humoral immune response to Treponema pallidum antigens in a homosexual man with asymptomatic human immunodeficiency virus infection.

The case presented here involves a 32-year-old homosexual man with human immunodeficiency virus (HIV) seropositivity and unusual manifestations of secondary syphilis. The patient presented with syphilitic keratoderma and chorioretinitis, and his appearance superficially resembled that of a patient with Reiter's syndrome. Although nontreponemal and treponemal tests for syphilis showed reactivity, the patient's humoral immune response to individual polypeptides of Treponema pallidum, measured by Western blot analysis, was markedly abnormal. The possible relationship between asymptomatic HIV infection and an abnormal humoral immune response to a second pathogen, in this case T. pallidum, is discussed. Our case is one of several recent cases of active syphilis reported in individuals with HIV seropositivity.

Adult↗

PubMed1988

Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp. pallidum.

Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp. pallidum (T. pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M. V. Norgard, N. R. Chamberlain, M. A. Swancutt, and M. S. Goldberg, Infect. Immun. 54:500-506, 1986). To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties. Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T. pallidum DNA fragment. A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system. Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular masses were observed that were identical to those previously detected on Western blots of native T. pallidum antigens with monoclonal antibodies. Sarkosyl extraction of E. coli recombinant cell envelopes localized the 47-kDa protein to both the inner and outer membranes of E. coli. The absolute requirement of detergents (N-lauroylsarcosine, 3-[(3-chloramidopropyl)dimethylammonio]-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40) for solubilization of the antigen from E. coli cell envelopes and the observation that the recombinant protein partitioned into the detergent phase on Triton X-114 solubilization were consistent with the fact that it is a hydrophobic, integral membrane protein. Western blots of the 47-kDa antigen purified by immunoaffinity chromatography supported results of previous reports that the 47-kDa protein is specific to pathogenic treponemes.

Antigens, Surface↗

PubMed1988

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.

Antigens, Bacterial↗

PubMed1988

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.

Animals↗

PubMed1987

Cloning and expression in Escherichia coli of the gene encoding the heat-modifiable major outer membrane protein of Haemophilus influenzae type b.

One major outer membrane protein (P1) of Haemophilus influenzae type b (Hib), with an apparent molecular weight of 34,000 (34K) as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), has been shown to be heat modifiable. After heating at 100 degrees C for 5 min in 2% SDS, the P1 protein exhibits an apparent molecular weight of 49,000 (49K) in SDS-PAGE. Monoclonal antibodies (MAbs) reactive with P1 bound to the surface of Hib, and one of these MAbs had a protective effect against the development of Hib bacteremia in an animal model for invasive Hib disease. A 6-kilobase Hib DNA insert containing the gene encoding this P1 protein was cloned into Escherichia coli by using the gamma gt11 expression vector. Recombinant phage expressing P1 were identified by screening phage plaques with a MAb directed against the P1 protein. Expression of the P1 protein by an E. coli lysogen carrying the recombinant phage was independent of both vegetative phage growth and induction of lacZ gene-directed transcription of the Hib DNA insert. The Hib DNA insert encoding the P1 protein was subcloned into the plasmid vector pBR322, and a transformant containing the recombinant plasmid pFRG100 was identified with the P1 protein-directed MAb in a colony blot-radioimmunoassay. Western blot (immunoblot) analysis determined that the recombinant P1 protein possessed heat-modifiability characteristics identical to those of the native Hib protein. The P1 protein was expressed on the surface of both the E. coli lysogen containing the recombinant phage and the E. coli transformant containing pFRG100. Western blot analysis of acute- and convalescent-phase sera from infants with Hib meningitis showed that antibodies in the convalescent-phase sera recognized the P1 protein expressed by the E. coli transformant containing pFRG100. The availability of this cloned Hib DNA insert encoding the Hib P1 protein and the expression of this protein on the surface of recombinant E. coli should facilitate the investigation of P1 for both its vaccinogenic potential and its functional role in the outer membrane of Hib.

Antibodies, Monoclonal↗

PubMed1987

Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D.

Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a protease-resistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.

Antigens, Bacterial↗

PubMed1987

Human IgG antibodies to Pseudomonas aeruginosa core lipopolysaccharide determinants are detected in chronic but not acute pseudomonas infection.

Human IgG response to Pseudomonas aeruginosa core lipopolysaccharide determinants was measured after both acute and chronic pseudomonas infection by using lipopolysaccharide purified from PAC605 cells (the most lipopolysaccharide-defective or "roughest" mutant of P. aeruginosa yet described) as a solid phase antigen in ELISA and Western blot immunoassay. The geometric mean IgG anti-PAC605 lipopolysaccharide titer of sera from 18 cystic fibrosis (CF) patients with chronic pseudomonas pulmonary infection was 1808, compared to 171 for convalescent sera of 10 patients with acute pseudomonas bacteremia (p less than 0.001) and 211 for 5 normal human volunteers (p less than 0.001). Western blot immunoassay demonstrated specific IgG anti-core antibodies in 11/18 sera from CF patients but not in the sera of the convalescent bacteremic patients or normal volunteers. IgG anti-Pseudomonas core lipopolysaccharide antibodies appear to be a marker of chronic infection; the possible protective role of these antibodies remains to be established.

Acute Disease↗

PubMed1986

The antigenic interrelationship between the endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum Nichols strain. I. Treponemicidal activity of cross-reactive endoflagellar antibodies against T. pallidum.

Treponemicidal activity against Treponema pallidum, Nichols strain, by anti-endoflagellar antibodies and the presence of antigenic interrelationships between the endoflagella of Treponema phagedenis biotype Reiter (TPR) and T. pallidum have been demonstrated. SDS-PAGE profiles of purified endoflagella from both organisms were similar, identifying five polypeptide bands for TPR (37,000, 33,000 doublet, 30,000, and 27,000 daltons) and five polypeptide bands for T. pallidum (35,000, 33,000 doublet, 30,000, and 27,000 daltons). Antiserum against TPR endoflagella identified identical bands on Western blots of TPR, T. pallidum, and the respective endoflagellar preparations. Western blots confirmed the presence of antibodies in normal human serum (NHS) against the 33,000 dalton treponemal endoflagellar proteins. The complement-dependent treponemicidal activity of NHS against T. pallidum was completely removed by absorption with purified TPR endoflagella. Furthermore, rabbit antisera against TPR endoflagella were reactive in the Treponema pallidum immobilization (TPI) test. These findings demonstrate that anti-endoflagellar antibodies are treponemicidal against T. pallidum. A possible mechanism for this activity is discussed in relation to the subsurface location of endoflagella.

Animals↗

PubMed1986

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain.

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.

Antibodies, Bacterial↗

PubMed1986

Serodiagnosis of syphilis by enzyme-linked immunosorbent assay with purified recombinant Treponema pallidum antigen 4D.

An enzyme-linked immunosorbent assay (ELISA) for syphilis has been developed that detects IgG antibody to purified recombinant Treponema pallidum surface antigen 4D. The 4D ELISA was capable of detecting 25 ng of 4D antigen-specific antibody. Neither 172 nonsyphilitic sera nor 20 false-positive sera in the Venereal Disease Research Laboratory test reacted in the 4D ELISA. The sensitivity of the 4D ELISA was comparable to that of the adsorbed fluorescent treponemal antibody test in primary, secondary, and latent disease. Most sera from patients with yaws or pinta were also reactive, a result indicating that a 4D antigen-like molecule also exists in the closely related pathogenic treponemes Treponema pertenue and Treponema carateum.

Antigens, Bacterial↗

PubMed1986

The surface of virulent Treponema pallidum: resistance to antibody binding in the absence of complement and surface association of recombinant antigen 4D.

The binding of immunoglobulin G present in syphilitic immune rabbit serum, syphilitic human serum, and rabbit antiserum to purified recombinant Treponema pallidum antigen 4D by T. pallidum, Nichols strain, was studied by immunoelectron microscopy. Treponemes were incubated with antiserum under the conditions of the T. pallidum immobilization test, in which T. pallidum-specific antibody renders the organism nonmotile and avirulent only in the presence of complement after a 16-h incubation period in an anaerobic environment. Antibody was not demonstrable on the surface of T. pallidum incubated with nonimmune rabbit serum or normal human serum in the presence of complement. Similarly, in the absence of complement, little or no antibody was found on the treponemal surface after incubation with syphilitic immune rabbit serum, syphilitic human serum, or rabbit antiserum directed against the recombinant 4D antigen. The addition of complement to syphilitic immune rabbit serum, syphilitic human serum, and anti-4D antibody resulted in immobilization and the deposition of antibody on the entire surface of the immobilized organisms. These results corroborate earlier work by other investigators demonstrating the resistance of freshly isolated T. pallidum to antibody binding in a variety of serological tests. Detection of 4D antigen on the surface of immobilized T. pallidum strongly implies that the use of T. pallidum immobilization test conditions provides a means to demonstrate the association of individual surface antigens on virulent T. pallidum. The resistance of T. pallidum to antibody binding may be relevant to the pathogenesis of syphilis.

Animals↗