Mucopolysaccharidase of Treponema pallidum

Treponema pallidum (Nichols strain) exhibited mucopolysaccharidase activity. Acidic mucopolysaccharides were broken down more rapidly by viable treponemes than by heat-inactivated treponemes or membrane filtrates of treponemal suspensions. Ouchterlony immunodiffusion demonstrated the occurrence of antibodies to the hyaluronidase-like enzyme within syphilitic sera. After intratesticular inoculation of 2 x 10(7) to 6 x 10(7) treponemes, these anti-mucopolysaccharidase antibodies were detected between 9 and 35 days postinoculation. In addition, acidic mucopolysaccharides were present in the serum of infected animals 9 and 16 days postinoculation. Immune serum that contained antibodies to the mucopolysaccharidase restricted treponemal breakdown of acidic mucopolysaccharides. It has been previously demonstrated that immune rabbit serum contains a factor that blocks attachment of T. pallidum (Nichols strain) to cultured mammalian cells. This factor was effectively absorbed by prior incubation with bovine hyaluronidase. It is postulated that T. pallidum attaches to acidic mucopolysaccharides on the surface of cultured cells through the mucopolysaccharidase enzyme at the surface of the organisms. These findings are discussed in terms of the histopathogenesis of T. pallidum with applications to the healing immune response.     

Antibody responses elicited against the Treponema pallidum repeat proteins differ during infection with different isolates of Treponema pallidum subsp. pallidum

Variation in the expression of the different Tpr proteins in the syphilis spirochete, Treponema pallidum subsp. pallidum, may have important implications in its ability to evade host immune detection and cause persistent infection. In the present study we examined the pattern of antibody responsiveness to different Tpr members during infection with three isolates of T. pallidum. There was variability in the specificities and temporal patterns of reactivity of the antibodies elicited against the individual Tpr proteins, suggesting that isolates may express different repertoires of Tpr proteins during infection.     

Surface Mucopolysaccharides of Treponema pallidum

The viscous mucoid fluid that accumulates within syphilitic lesions may be due to breakdown of host tissue during infection, or may be synthesized by Treponema pallidum. Experiments were performed to investigate the acidic mucopolysaccharides that occur at the surface of T. pallidum (Nichols strain). These mucopolysaccharides were demonstrated by reaction with acidified bovine serum albumin and by agglutination with wheat germ agglutinin and soybean agglutinin. The polycations ruthenium red and toluidine blue influenced treponemal survival. Concentrations of both compounds at 200 mug/ml inhibited survival, whereas concentrations at 0.1mug/ml enhanced survival. The mucopolysaccharide concentration within the mucoid fluid that accumulates during intratesticular infection was determined by reaction with acidified bovine serum albumin; it ranged from 10,000 mug/ml to less than 8 mug/ml. The addition of this mucoid fluid to treponemal suspensions resulted in differing effects on T. pallidum survival. Some preparations were inhibitory, and others were stimulatory. Commercial preparations of hyaluronic acid and chondroitin sulfate at 400, 200, 100, and 50 mug/ml were detrimental to treponemal survival. The organisms exhibited pronounced clumping in the presence of the higher concentrations of hyaluronic acid. These clumps of treponemes were comprised of mucopolysaccharides as shown by acidified bovine serum albumin and toluidine blue reactions and by hyaluronidase degradation. Results are discussed in terms of the derivation and potential role of acidic mucopolysaccharides at the surface of T. pallidum.     

Immunoglobulin Class and Subclass Distribution of Dextran-Reactive Antibodies in Human Reactors and Non Reactors to Clinical Dextran

The red cell-linked antigen-antiglobulin reaction (RCLAAR) with stearoyldextran-coated erythrocytes was used to characterize the immunoglobulin (Ig) classes and IgG subclasses of dextran reactive antibodies (DRA) in 27 dextran reactors (DR) and 96 on reactors (DNR). High titres of dextran reactive IgG were regularly found in sera of patients with severe dextran-induced anaphylactoid/anaphylactic reactions (DIAR) prior to the infusion. In four lethal cases IgG antibodies were found to be in the highest titre range of 16,384 to 32,768. In addition, high IgA and IgM titres were also in severe DIAR. DNR had much lower titres of dextran reactive antibodies of IgG, IgM and IgA classes and IgD antibody was absent in both groups. Dextran reactive IgE antibodies were not demonstrable in DR. Dextran reactive IgG2, IgG3, IgG4 and IgG1 (indirect measurement) were demonstrated in both DR and FNR. Dextran infusion caused variable neutralization in all Ig classes and IgG subclasses, but the contribution of IgG2 was considered most important because of its high titres and most pronounced neutralization in severe DIAR. It is concluded that DRA mainly of the IgG class, play a critical pathogenic role in the induction of severe DIAR, which accordingly is classified as immune complex (Type III) anaphylaxis. The method of RCLAAR allows to delineate a risk group of about 2% of potential reactors.     

Surface Characterization of Virulent Treponema pallidum

Characterization of the surface of Treponema pallidum was accomplished by [¹²⁵I]lactoperoxidase-catalyzed iodination of intact organisms and sensitive radioimmunoprecipitation and gel electrophoresis technology. At least 11 outer membrane proteins with molecular weights ranging from 89,000 (89K) to 20K were identified, and all elicited high titers of antibody in experimentally infected rabbits. Proteins of 89.5K, 29.5K, and 25.5K previously implicated as ligands involved in attachment (J. B. Baseman and E. C. Hayes, J. Exp. Med. 151:573-586, 1980) were found to reside on the treponemal surface. Low levels of the 89.5K treponemal protein were released by high salt concentrations, whereas the remaining comigrating material was neither radioiodinated nor released with selective detergents. Other lower-molecular-weight (60K, 45K, and 30K) surface proteins were extracted with octyl glucoside detergent, suggesting their hydrophobic interaction with the external membrane. The molecular organization of surface proteins was studied by employing the cross-linker dithiobis(succinimidyl)-propionate, and data suggested the presence of a highly fluid envelope resulting in random collisions by the surface proteins. The biological function of the treponemal outer envelope proteins was evaluated using, as the indicator system, adherence of T. pallidum to monolayer cultures of eucaryotic cells. Trypsin treatment of motile, freshly harvested organisms decreased the extent of surface parasitism to normal rabbit testicular cells, reinforcing the idea of the proteinaceous nature and role of treponemal ligands for attachment. Other data supported functional and antigenic relatedness among the implicated ligands. Finally, brief periodate treatment of human epithelial (HEp-2) and normal rat testicular cells as well as casein-elicited rabbit peritoneal macrophages significantly reduced the extent of treponemal parasitism, suggesting a role of specific host membrane molecules as mediators of attachment.     

Outer Envelope of Virulent Treponema pallidum

Ultrathin sections of virulent Treponema pallidum (Nichols strain) were examined with the electron microscope, and the presence of an outer cell envelope was documented.     

Lipids of Treponema pallidum Kazan 5

The lipid composition of Treponema pallidum Kazan 5 cultivated in a lipid-defined medium was investigated. Lipids comprised 18 to 20% of the dry weight of the treponeme. Glycolipid and phospholipids accounted for 90 to 95% of the total lipids and free fatty acids made up the remaining 5 to 10%. The major polar lipids were the glycolipid, 1-(O-alpha-d-galactopyranosyl)-2,3-diglyceride (45 to 55%), and phosphatidylcholine (30 to 40%). Phosphatidylethanolamine (5 to 10%), an unidentified compound (1 to 2%), and occasional trace amounts of diphosphatidylglycerol (cardiolipin) were also found. The monogalactosyldiglyceride was also a major component (50%) of the lipids of the Reiter, Noguchi, and Nichols strains of T. pallidum. The fatty acid composition of Kazan 5 usually consisted of saturated and unsaturated fatty acids ranging from 14 to 18 carbons depending upon the fatty acids added to the culture medium. When the cells were cultivated on elaidic acid (trans-9-octadecenoic acid), their lipids contained only elaidic acid.     

Molecular differentiation of Treponema pallidum subspecies

Treponema pallidum includes three subspecies of antigenically highly related treponemes. These organisms cause clinically distinct diseases and cannot be distinguished by any existing test. In this report, genetic signatures are identified in two tpr genes which, in combination with the previously published signature in the 5' flanking region of the tpp15 gene, can differentiate the T. pallidum subspecies, as well as a simian treponeme.     

Laboratory Evaluation of Three Rapid Diagnostic Tests for Dual Detection of HIV and Treponema pallidum Antibodies

The performance of three research-use-only, dual HIV and syphilis rapid diagnostic tests (RDTs) was evaluated for 150 patient serum samples and compared to reference HIV and Treponema pallidum antibody detection methods. The RDTs performed comparably, with sensitivities of 93 to 99% and specificities of 97 to 100%. The kappa statistic between the RDTs was 0.95.     

Performance Evaluation of the Elecsys Syphilis Assay for the Detection of Total Antibodies to Treponema pallidum

Syphilis is a health problem of increasing incidence in recent years that may have severe complications if not diagnosed and treated at an early stage. There are many diagnostic tests available for syphilis, but there is no gold standard, and diagnosis therefore usually relies upon a combination of tests. In this multicenter study, we evaluated the treponemal Elecsys syphilis assay for use in the diagnosis of syphilis in routine samples, i.e., when syphilis is suspected or during antenatal or blood donation screening. The sensitivity and specificity of the Elecsys syphilis assay were compared head to head with those of other treponemal assays used in routine clinical practice and were assessed in potentially cross-reactive samples from patients with Epstein-Barr virus, HIV, and Lyme disease. In a total of 8,063 syphilis-negative samples collected from routine diagnostic requests and blood donations, the Elecsys syphilis assay had a specificity of 99.88%. In 928 samples previously identified as syphilis positive, the sensitivity was 99.57 to 100% (the result is presented as a range depending on whether four initially indeterminate samples are included in the assessment). The specificity of the Elecsys syphilis assay in patients with other infections was 100%; no false-positive samples were identified.     

Erythema Multiforme Caused by Treponema pallidum in a Young Patient with Human Immunodeficiency Virus Infection

Erythema multiforme (EM) is usually caused by drug reactions or virus infection. We report a case of secondary syphilis presenting as EM in an HIV-infected patient, proved by immunohistochemical staining, which is rare in the literature. It is valuable to determine the etiology of EM to optimize treatment.     

Immunoglobulin Class of Sperm Antibodies in Cervical Mucus From Infertile Women

ABSTRACT: By immunoaffinity chromatography using anti-IgG, anti-IgM, and anti-IgA coupled to CNBr-activated sepharose 4B, the immunoglobulin class of sperm-agglutinating antibodies was investigated in cervical mucus from four infertile women. In all patients, it was found that the sperm antibodies in cervical mucus belonged to the IgA class, whereas in serum, which was studied in two of the patients, IgG sperm antibodies were demonstrated. Absorption of the four cervical mucus samples with anti-secretory component sepharose 4B revealed that the IgA antibodies in at least two of the samples were SC-IgA antibodies. Investigation of a third sample by sucrose gradient ultracentrifugation revealed that the IgA sperm antibodies were characterized by a sedimentation coefficient between 9S and 13S, strongly suggesting the presence of SC-IgA antibodies. Accordingly, the sperm-agglutinating antibodies were SC-IgA antibodies in at least three of the four samples studied.     

Characterization of Two Murine Monoclonal Antibodies Reactive with Human B Cells

We describe two monoclonal antibodies, HH1 and HH2. Both reacted selectively with surface immunoglobulin (sIg)-positive human B cells. Both antibodies stained on average 7-8% of peripheral blood mononuclear cells. They have not been found to react with cells or cell lines of other haematopoietic cell lineages, except that HH2 was positive on a small percentage of cells of the erythroid cell line K562. The molecular weight of the HH1 antigen was 95 kD, as established by Western blotting. Neither of these two antibodies reacted with Ig determinants, Fc receptors, complement receptors, or known class-I or class-II molecules. A combination of these antibodies was used in a direct panning technique for high-yield enrichment of normal B lymphocytes from peripheral blood. The enriched B cells could be further purified by lysis of T cells (final yield, on average 72 +/- 8% of initial B cells) or by a second panning (yield, 35 +/- 11%). The purified B cells contained less than 1% contaminating T cells and less than 0.5% monocytes and were used in an assay for B-cell-stimulating factor which they showed a normal and very reproducible proliferative response.     

Isolation and characterization of antigens from Treponema phagedenis biotype Reiter cross-reacting with Treponema pallidum

The specific aims of the studies reviewed here were to design a rational purification strategy for Reiter treponeme antigens using combinations of different chromatographic principles, to isolate and characterize the Reiter treponemal antigens, especially the antigens labelled TR-b, TR-c, TR-dl, TR-d2, and TR-e cross-reacting with antigens in Treponema pallidum, to develop and evaluate the use of these purified antigens in syphilis diagnostic enzyme-linked immunosorbent assays (ELISA), to investigate whether Reiter treponeme antigens can induce protective immunity against experimental syphilis in rabbits. By combining anion exchange chromatography, gel filtration, agarose gel electrophoresis, hydrophobic interaction chromatography, and affinity chromatography on Iminodiacetic acid-Sepharose CL 4B and Lysine Sepharose 4B we were able to isolate seven different water soluble Reiter antigens from one single Reiter sonicate supernatant (I, II, III, IV, V, VI). The applied chromatographic matrices were selected due to their efficiency for separation of the Reiter antigens in pilot experiments. In addition to the above mentioned Reiter antigens additional antigens labelled TR-o (V) and LPS (VI) were isolated. TR-b, TR-c, and TR-o were shown to be protein antigens (III, IV, V). The TR-c antigen of the Reiter treponeme cross-reacted not only with an antigen in T. pallidum but also with an antigen common to a wide range of bacteria (IX). The TR-d antigen composed of a ribonucleic acid component (TR-dl) (II) and a protein component (TR-d2). The TR-e antigen represented the flagellum of the bacterium (I), and the LPS antigen was a pure lipopolysaccharide antigen (VI).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect macrophage activation on infection with Treponema pallidum.

An in vitro method is described to measure the inhibitory activity of murine peritoneal exudate cells against viral plaque formation by a bovine herpes-virus-infectious bovin'e rhinotracheitis virus. Microtiter plates containing 96 bovine kidney cell monolayers were infected with a range of virus concentration and peritoneal exudate cells were subsequently added. When a sufficient number of cells was added, viral plaques were not detectable and free infectious virus did not occur in the culture fluids. The inhibitory cell type adhered to glass and was presumably a macrohage. Although inhibitory of viral plaques was presumably a macrophage. Although inhibition of viral plaques was complete and free virus could not be detected, virus was not eliminated from the monolayers since on removal of cells, the degree of virus cytopathology and yield of virus after a further 48 h of incubation was the same as in 48-h infected control monolayers. The significance of peritoneal exudate-cells-induced virus suppression as a model to understand herpesvirus latency is briefly discussed.?Author     

Runting syndrome in neonatal rabbits infected with Treponema pallidum

Infection of rabbits with Treponema pallidum prior to the 2nd week of life usually resulted in progressive runting leading to death about the 7th–13th week of age. The severity of the disease could be evaluated at any particular time by the percentage weight retardation. In this small series there was a suggestion that the runting index (R.I.) and the presence or absence of reagins might correlate with the prognosis of the disease. Weight retardation of more than 25% and a R.I. of less than 0·81 were usually associated with irreversible, pathological processes refractory to penicillin therapy.

The disease occurred in degrees of severity. There was a mild form with a R.I. of about 0·90, reactive Malpighian corpuscles and increased number of histiocytic like cells in the spleen but with normal thymuses. A moderate form was described with a R.I. of about 0·81 and partial depletion of lymphocytes in the spleen with predominance of histiocytic like cells. The thymuses were normal. A severe form was characterized by a R.I. of 0·65–0·76, depletion of lymphocytes and increased number of histiocytic like cells in both spleen and thymus.

The possibility that the syphilitic runting syndrome in baby rabbits is the result of a direct, or indirect action of the treponemes on the lymphatic system is advanced.

     

Phagocytosis of opsonized Treponema pallidum subsp. pallidum proceeds slowly.

Macrophages were found to phagocytize Treponema pallidum subsp. pallidum attached to polycarbonate filters. This environment simulated the in vivo interaction of surface-adherent treponemes with macrophages. The phagocytosis of T. pallidum subsp. pallidum was found to proceed slowly. Heat-killed T. pallidum subsp. pallidum were susceptible to opsonization with 2% immune serum, whereas live treponemes were resistant to this concentration of antibody. High concentrations of immune serum were found to increase phagocytosis of the spirochetes. Live T. pallidum subsp. pallidum had bound limited quantities of immunoglobulin G in vivo, and only opsonization with 20% immune serum resulted in a detectable increase in surface-bound immunoglobulin in vitro. Kinetic studies suggested a steady rate of phagocytosis that is considerably slower than with other bacteria. Scanning electron microscopy studies of the phagocytizing macrophages showed that the treponemes were detached from the membrane filters and scooped onto the ruffled portion of the macrophage surface. This lengthy physical process, along with the lack of a dramatic increase in ingestion after opsonization, may account for the slow rate of phagocytosis.     

Molecular basis of immunological cross-reactivity between Treponema pallidum and Treponema pertenue

Protein antigens of Treponema pallidum, Nichols strain, and Treponema pertenue, Gauthier strain, were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Treponemal proteins were solubilized in 1% sodium dodecyl sulfate, electrophoresed on 12.5% polyacrylamide gels, and either stained with Coomassie brilliant blue or electrophoretically transferred to nitrocellulose paper. These antigen blots were incubated with sera from rabbits infected with either T. pallidum or T. pertenue and 125I-labeled staphylococcal protein A and exposed to X-ray film for visualization of antigenic molecules. Protein profiles of each organism separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue showed no distinguishable differences. Antigenic profiles as determined by Western blots were similar with two exceptions. A 39,500-dalton band was present on T. pertenue but absent from T. pallidum, and a 19,000-dalton band was present on T. pallidum but absent from T. pertenue (although two additional antigenic bands at 21,000 and 18,000 daltons were seen on T. pertenue). Because these differences were detected by using antisera raised against either T. pallidum or T. pertenue, these molecules must contain some antigenic determinants in common despite their differences in molecular weight.