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Characterization of the humoral immune response of the rabbit to antigens of Treponema pallidum after experimental infection and therapy.

The development of IgM and IgG antibody responses to antigenic molecules of Treponema pallidum was examined in intratesticularly and intradermally infected rabbits by use of immunofluorescence (IF) and Western blotting techniques. IgM antibody was first detectable on day 6 following intratesticular infection and reached maximal IF titers during the period of clinical orchitis (days 10-17). IgG reached peak IF titers in the resolution period following clearance of most organisms from the testes (after day 17). Initial IgM antibody responses on day 6 were directed against the 46-, 43-, and 37-kdalton molecules, with reactivity to the 14- and 12-kdalton molecules appearing on day 10. IgG antibody, which was first apparent in the Western blot on day 6, was directed against the 37-kdalton molecule. IgG antibody to the 46-kdalton molecule was first apparent on day 13, and the full spectrum of 22 molecules was recognized on day 31. Curative therapy of intradermally infected rabbits one week after infection resulted in lower IgG antibody titers but did not abrogate the development of humoral responses to all of the major antigens.

Animals↗

Serum requirement for the multiplication of Treponema pallidum in a tissue-culture system: association of growth-promoting activity with the protein fraction.

The nature of the serum requirement of Treponema pallidum subspecies pallidum (Nichols strain) was examined in a culture system utilizing Sf1Ep cottontail rabbit cells. In this system, significant multiplication of treponemes occurs in the presence of select lots of fetal bovine serum (FBS) or calf serum (CS) at concentrations of greater than or equal to 5% (vol/vol). Heat-inactivation of the serum greatly enhances treponemal multiplication, and normal human serum was found to be as effective as FBS in supporting the growth of T. pallidum. The protein fraction of FBS obtained by membrane ultrafiltration was capable of supporting the multiplication of T. pallidum when added to the basal tissue culture medium; an average increase of 23-fold was observed in these cultures, as compared with a mean increase of 25-fold in the 20% FBS controls. In contrast, the ultrafiltrate fraction of FBS (consisting of compounds with molecular weights of less than 10,000 daltons) did not support either growth or the retention of motility. Proteins precipitable with 25% (wt/vol) polyethylene glycol (i.e., albumin, transferrin, ceruloplasmin, and other proteins) also promoted the growth of T. pallidum. This observation provides further evidence that the required serum components are associated with the protein fraction.

Animals↗

IgG and IgM antibody reactivity to antigens of Treponema pallidum after treatment of syphilis.

The persistence or loss of IgG and IgM antibody specificities for individual polypeptides of Treponema pallidum after therapy for syphilis was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by the Western blot technique. Both IgG and IgM antibodies to as many as 12 treponemal antigens, including a major 47-kdalton molecule, were evident in plasma from patients with untreated primary syphilis. IgM reactivity declined rapidly and uniformly after therapy, whereas IgG persisted despite some diminution in intensity of staining. Faint-to-moderate IgM and strong IgG antibody reactivities to at least 22 treponemal antigens (12-85 kdaltons) were identified in plasma from patients with untreated secondary and early latent syphilis. Again, IgG antibody declined slightly in staining intensity after treatment but continued to show reactivity against all molecules detected initially. IgM antibody reactivity declined more rapidly and was lost entirely against some determinants, including the 14- and 12-kdalton molecules. Immunofluorescence titers of IgG and IgM antibodies to T. pallidum in sera from these patients generally correlated with results of Western blot analysis. Antibody to the 12-, 14-, and 47-kdalton molecules of T. pallidum may have potential diagnostic applications.

Animals↗

Immunochemical characterization and purification of Treponema pallidum antigen TpD expressed by Escherichia coli K12.

The immunochemical properties of the Treponema pallidum antigen TpD, as expressed by Escherichia coli K12, was investigated by crossed immunoelectrophoresis in which an affinity-purified antibody to this antigen was used. Two immunologically cross-reacting components of TpD with different mobility were demonstrated. Affinity-purified antibodies were used in obtaining purified TpD and in determining the cellular localization of TpD in T. pallidum by immunoelectron microscopy. TpD was localized on the surface of methanol-fixed T. pallidum. Twenty sera from patients with secondary syphilis and 20 sera from nonsyphilitics were examined in crossed immunoelectrophoresis. All sera from patients with secondary syphilis and none from nonsyphilitics contained antibodies to the TpD components. Because TpD seems to be surface associated and a major immunogen during infection with T. pallidum, this antigen might be useful for development of a vaccine against syphilis and for development of improved methods for serodiagnosis of syphilis.

Antibodies, Bacterial↗

Demonstration of Treponema pallidum in axons of cutaneous nerves in experimental chancres of rabbits.

After cutaneous inoculation of viable Treponema pallidum subsp pallidum into the skin of chancre-immune or previously uninfected rabbits, organisms move from perivascular connective tissue to localize extracellularly in hair follicles, erector pili muscles, and cutaneous nerves. Large numbers of intact organisms can be seen within the perineurium using electron microscopy, and after extensive sampling a few organisms can be detected within the axon cytoplasm. These findings support the concept that T. pallidum may be able to pass along the nerve fibers to the central nervous system.

Animals↗

Enzyme-linked immunospot assay for the diagnosis of active Treponema pallidum infection during the various stages of syphilis.

BACKGROUND: Specific serologic assays for syphilis cannot differentiate current infections from past infections and are inefficient to monitor efficacy of antibiotic therapy. GOAL: To develop a new immunologic assay for the identification of active Treponema pallidum infection during the various stages of syphilis. STUDY DESIGN: Peripheral blood mononuclear cells obtained from patients with syphilis in an STD clinic were tested for T. pallidum-specific circulating antibody-secreting cells (ASC) by an enzyme-linked immunospot assay (ELISPOT). RESULTS: Specific ASC were demonstrated in all six patients with primary syphilis and in 14 of 16 patients diagnosed with secondary syphilis. ASCs were undetectable in five patients 8 to 16 days after appropriate therapy, but persisted in one case that was considered treatment failure. Among the 13 patients diagnosed with latent syphilis, six (46%) demonstrated ASC, reflecting antigenic stimulation. CONCLUSION: The ELISPOT assay is effective for the diagnosis of primary and secondary syphilis. The presence of circulating ASC suggests persistent active infection in some patients during the latent disease stage.

Adolescent↗

Epidemiology of human immunodeficiency virus, viral hepatitis (B and C), treponema pallidum, and human T-cell lymphotropic I/II virus among men who have sex with men in Buenos Aires, Argentina.

BACKGROUND: Human immunodeficiency virus (HIV) and sexually transmitted infections (STI) are prevalent among men who have sex with men (MSM). GOAL: To estimate the prevalence of HIV and STIs in this group. STUDY: A total of 694 MSM were tested for HIV, hepatitis B (HBV), hepatitis C (HCV), human T-cell lymphotropic (HTLV-I/II) viruses and Treponema pallidum infection. RESULTS: HIV, HBV, and T pallidum were detected in 13.8%, 37.7%, and 16.9% of subjects, respectively. Prevalences of 1.9% and 0.3% were detected for HCV and HTLV-I/II. A prior history of STI was the most predictor for HIV, HBV, and T pallidum. Use of illegal drugs, blood transfusion history, and multiple sexual partners were associated with HCV. The 2 most common co-infections were HBV/T pallidum and HIV/HBV. CONCLUSIONS: Infection with HIV, HBV, and T pallidum was elevated among MSM. Routine testing, education, vaccine-based prevention, and control programs need to be implemented in this high-risk population.

Adolescent↗

Treponema amylovorum sp. nov., a saccharolytic spirochete of medium size isolated from an advanced human periodontal lesion.

A highly motile, medium-size, saccharolytic spirochete was isolated from an advanced human periodontal lesion in medium OMIZ-Pat supplemented with 1% human serum. The growth of this organism is dependent on either glucose, maltose, starch, or glycogen. The cells contain six endoflagella, three per pole, which overlap in the central region of the cell body. On the basis of its cell morphology and enzyme activities, as well as its sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein and antigen profiles, this organism is clearly distinct from all previously cultured spirochetes. The presence of a novel species is supported by the 16S rRNA sequence of this organism, which places it in phylotype 19 of Choi et al. (B. K. Choi, B. J. Paster, F. E. Dewhirst, and U. B. Göbel, Infect. Immun. 62:1889-1895, 1994). The only isolate, strain HA2P, is designated the type strain of a novel species, for which we propose the name Treponema amylovorum.

Bacterial Proteins↗

Molecular cloning of a gene (poIA) coding for an unusual DNA polymerase I from Treponema pallidum.

The gene coding for the DNA polymerase I from Treponema pallidum, Nichols strain, was cloned and sequenced. Depending on which of the two alternative initiation codons was used, the protein was either 997 or 1015 amino acids long and the predicted protein had a molecular mass of either 112 or 114 kDa. Sequence comparisons with other polA genes showed that all three domains expected in the DNA polymerase I class of enzymes were present in the protein (5'-3' exonuclease, 3'-5' exonuclease and polymerase domains). Additionally, there were four unique insertions of 20-30 amino acids each, not seen in other DNA polymerase I enzymes. Two of the inserts were near the boundary of the two exonuclease domains and the other two interrupted the 3'-5' exonuclease domain which is involved in proofreading. The predicted amino-acid sequence had an exceptionally high content of cysteine (2.4% compared with <0.05% for most other sequenced DNA polymerase I enzymes). The polA gene was further cloned into pProEXHTa for expression and purification. The transformants expressed a protein of 115 kDa. Antibodies raised against synthetic peptide fragments of the putative DNA polymerase I recognised the 115-kda band in Western blot analysis. No DNA synthesis activity could be demonstrated on a primed single-stranded template. Although significant quantities of the protein were produced in the host Escherichia coli carrying the plasmid, it was not capable of complementing a polA(-) mutant in the replication of a polA-dependent plasmid.

Amino Acid Sequence↗

The effect of reducing and other agents on the motility of Treponema pallidum in an acellular culture medium.

The maintenance of Treponema pallidum motility was investigated in an acellular medium based on T. pallidum immobilization test medium. The acellular medium contained cysteine, glutathione, thioglycollate and dithiothreitol as reducing agents and had a redox potential of -275 +/- 25 mV at pH 7.3. In an atomosphere containing 3% O2, motile treponemes survived four times longer when calf serum and bovine serum albumin were added to the medium. The selective omission of glutathione and, particularly, thioglycollate prolonged the survival of motile treponemes almost fivefold. In addition, stored medium, in which thioglycollate had become inactive, sustained motile treponemes for longer than did freshly prepared medium. Thus, thioglycollate is toxic for the organisms. It may be omitted from the medium because low redox potentials can be achieved without it.

Culture Media↗

Sterol requirement for the growth of Treponema hyodysenteriae.

The addition of cholesterol to a liquid medium containing bovine serum albumin (BSA) fraction V or acetone-delipidized BSA fraction V instead of serum stimulated the growth of Treponema hyodysenteriae, a serum-requiring spirochaete associated with swine dysentery. As little as 1.25 micrograms cholesterol ml-1 increased viable counts about 1000-fold. Sitosterol and cholestanol, but not pregnenalone, cholestenone or stigmasteriol, produced a growth response comparable to that of cholesterol. The results suggest that T. hyodysenteriae requires a sterol for growth.

Cholesterol↗

Avoidance of host defences by Treponema pallidum in situ and on extraction from infected rabbit testes.

Electron microscopic observations of acutely infected rabbit testes showed that the majority of Treponema pallidum were extracellular, and confined to interstitial regions of the tissue. Organisms were often adjacent to small blood vessels, where they should be freely accessible to non-specific humoral and cellular defence mechanisms. However, there was no accumulation of leucocytes in blood vessels or infiltration of inflammatory cells into infected areas. Inoculation of live treponemes into perforated plastic chambers which had been implanted subcutaneously in rabbits or guinea-pigs did not incite significant infiltration into the chamber fluid of inflammatory cells, in contrast to that seen after inoculation of Neisseria gonorrhoeae. Interaction of antibody from infected rabbits with live treponemes freshly extracted from rabbit testes could not be detected by an indirect fluorescent antibody method. These observations suggest that T., pallidum escapes recognition by host defences.

Animals↗

The outer membrane of Treponema pallidum: biological significance and biochemical properties.

Rabbits infected intravenously with Treponema pallidum were not markedly febrile, and the pyrogenicity of treponeme preparations administered intravenously to rabbits was negligible. The antibiotic polymyxin B did not induce any ultrastructural changes on the treponemal surface and was not lethal (immobilizing) for T. pallidum, which was, however, highly susceptible to detergents such as SDS. Extraction of treponemes with Triton X-100 removed the outer membrane (despite the presence of Mg2+) as shown by electron microscopy, and solubilized a limited number of proteins detectable by SDS-PAGE, including a dominant antigen (47 kDal) demonstrated by immunoblotting. None of the proteins were heat-modifiable. Periodic acid-silver staining of polyacrylamide gels for carbohydrate together with protease K digestion did not demonstrate major carbohydrate components in whole treponemes, or in the Triton-soluble fraction. Surface iodination of intact treponemes revealed very little surface exposure of treponemal proteins, although a protein which co-migrated with host albumin was labelled and appeared to be associated with the treponemal surface. Many treponemal proteins were, however, labelled when iodination was done in the presence of Triton. These observations, indicate that the outer membrane of T. pallidum differs significantly from those of many Gram-negative pathogens.

Animals↗

Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12.

A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the 'common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.

Antigens, Bacterial↗

Monoclonal antibodies directed against surface-associated polypeptides of Treponema pallidum define a biologically active antigen.

Murine monoclonal antibodies (Mabs) were raised against two outer-membrane-associated polypeptides of Treponema pallidum (47 and 44 kDa). Three Mabs against each polypeptide were investigated further and only those directed against the 44 kDa polypeptide were demonstrated to have immobilizing activity. The specificity of the Mabs for T. pallidum was determined by Western blotting procedures and the surface association of the antigens was inferred by immunogold electron microscopy. The clear distinction between these two polypeptides in their biological activity could help to explain the pathobiology of syphilis infections as the 47 kDa antigen has been shown to be associated with the outer membrane of this organism. Inactivity of such a surface-located protein in antibody-mediated anti-treponemal mechanisms could account for the observed ability of this organism to survive in the face of strong antibody responses in infection.

Animals↗

Association of Treponema hyodysenteriae with porcine intestinal mucosa.

The association of Treponema hyodysenteriae with porcine caecal and colonic mucosal surfaces was studied by electron microscopy after orogastric inoculation of pigs with pure cultures. Examination of caecal and colonic mucosa from infected and control animals revealed that large numbers of the spirochaete were associated only with intestinal mucosal surfaces of infected animals. Further examination of the intestinal mucosa from infected pigs showed that T. hyodysenteriae colonized two sites preferentially: the mucus-filled crypts of Lieberkühn and the mucus gel covering the epithelium. Furthermore, no evidence of either specific or nonspecific adhesion to the epithelium proper was found, suggesting that penetration of, or trapping in the mucus gel may be the predominant mechanism of mucosal association by T. hyodysenteriae. Moreover, T. hyodysenteriae was also observed to be highly motile in intestinal mucus, moving faster than any other organism present, and this 'high speed' motility appeared to facilitate penetration into the mucosa. The pattern of motility observed was also highly suggestive of chemotaxis, and this was subsequently confirmed using an in vitro assay to porcine mucus material. It is suggested, therefore, that motility and chemotaxis are important factors/mechanisms in the association and colonization of porcine intestinal mucosa by T. hyodysenteriae.

Animals↗

Antigenic and structural analysis of Treponema denticola.

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determined by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product of a larger, heat-modifiable polypeptide. Based on the results of SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a 'rough' lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology of the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath and core structure and polypeptide composition characteristic of that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure of antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detailed molecular analysis of the ultrastructure and antigenicity of T. denticola.

Antigens, Bacterial↗