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PubMed1997

Nucleotide sequence of the Treponema pallidum eno gene.

We determined the nucleotide sequence of the enolase (eno) gene of Treponema pallidum, the noncultivable agent of syphilis. The deduced amino acid sequence of T. pallidum enolase (Eno) is 432 amino acids long with a predicted molecular mass of 46.7 kDa. The Eno amino acid sequence has a high degree of homology to the amino acid sequences of prokaryotic and eukaryotic Eno. This is the first eno sequence reported for a bacterium in the order Spirochaetales.

Amino Acid Sequence↗
PubMed1997

Identification, sequences, and expression of Treponema pallidum chemotaxis genes.

Treponema pallidum, the agent of syphilis, is a pathogenic spirochete that has no known mechanisms of genetic exchange and cannot be continuously cultivated in vitro. A probe based on the nucleotide sequence of the T. pallidum cheA gene was used to screen a T. pallidum genomic DNA library. A treponemal DNA region containing four open reading frames (orfs) was identified. The proteins encoded by these orfs have significant homology with proteins involved in bacterial chemotaxis. The orfs have been designated cheA, cheW, cheX, and cheY. The cheA, cheW, and cheY genes were individually-cloned and expressed in vitro. The observed molecular mass of each protein correlated well with its predicted molecular mass. Reverse transcriptase-PCR data indicate that cheA through cheY are co-transcribed. The organization of these genes suggests that they comprise an operon. We hypothesize that the ability to sense and respond to nutrient gradients is important for the survival and dissemination of T. pallidum in vivo. The presence of a putative che operon strongly suggests that T. pallidum has the potential for a chemotactic response.

Amino Acid Sequence↗
PubMed1983

Lack of endotoxin in Borrelia hispanica and Treponema pallidum.

Borrelia hispanica from infected guinea pigs and Treponema pallidum from testicular syphilomas of rabbits were assayed for the presence of endotoxin with the Limulus lysate test. A suspension of Borrelia, containing 1.3 X 10(8) spirochetes/ml, was nonreactive both when it was tested as intact organisms, and when tested after disruption of the spirochetes by sonication. Eight different suspensions of treponemes, ranging from 0.6 X 10(9) to 3 X 10(9) treponemes/ml, were negative at a 1:10 dilution and were no more active than control suspensions of normal rabbit testes. Therefore, it was concluded that T. pallidum, as well as the Borrelia, possessed no endotoxin.

Animals↗
PubMedDate not supplied

Testing umbilical cords for funisitis due to Treponema pallidum infection, Bolivia.

To establish the frequency of necrotizing funisitis in congenital syphilis, we conducted a prospective descriptive study of maternal syphilis in Bolivia by testing 1,559 women at delivery with rapid plasma reagin (RPR). We examined umbilical cords of 66 infants whose mothers had positive RPR and fluorescent treponemal antibody absorption tests. Histologic abnormalities were detected in 28 (42%) umbilical cords (seven [11%] had necrotizing funisitis with spirochetes; three [4%] had marked funisitis without necrosis; and 18 [27%] had mild funisitis), and 38 [58%] were normal. Of 22 umbilical cords of infants from mothers without syphilis (controls), only two (9%) showed mild funisitis; the others were normal. Testing umbilical cords by using immunohistochemistry is a research tool that can establish the frequency of funisitis due to Treponema pallidum infection.

Antibodies, Bacterial↗
PubMed1989

Changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum in syphilis patients after treatment.

The changes of serum IgG antibody reactivity to protein antigens of Treponema pallidum after treatment of syphilis were observed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Until 9 to 12 months after treatment, it was seen that there was a loss of several antibodies and some diminution in their reactivity in primary, secondary and early latent syphilis, but no changes occurred in late latent and reinfected syphilis. In primary syphilis, there was a significant loss of two IgG antibodies to the treponemal antigens of molecular weights 68,500 and 47,000 at 11 months after treatment. According to our previous study, the treponemal antigen of molecular weight 68,500 was T. pallidum specific and appeared only in primary syphilis, and that of molecular weight 47,000 was one of the major antigens of T. pallidum. The reaction between serum IgG antibodies of 14 patients who had been treated for secondary, early latent and late latent syphilis 2 to 14 years ago and major antigens of T. pallidum was observed and any loss or decrease in reactivity was not discovered. From the results obtained, it was concluded that the observation of serum IgG antibody reactivity to protein antigens of T. pallidum is not helpful in evaluating the efficacy of treatment in secondary, early latent, late latent and reinfected syphilis. However, serum IgG antibodies to treponemal antigens of molecular weights 68,500 and 47,000 could possibly be useful in the assessment of the efficacy of treatment in primary syphilis.

Antibodies, Bacterial↗
PubMed1991

Detection of Treponema pallidum in tissue: a comparative study of the avidin-biotin-peroxidase complex, indirect immunoperoxidase, FTA-ABS complement techniques and the darkfield method.

With 37 formalin-fixed, paraffin embedded specimens from the lesions of 30 patients with primary, secondary or gastric syphilis, we performed avidin-biotin-peroxidase complex (ABC), indirect immunoperoxidase (IIP) and FTA-ABS complement techniques. Darkfield examination was done in 17 skin lesions. The immunoperoxidase technique, especially the ABC technique, revealed higher reactivity than the FTA-ABS complement technique and darkfield examination in detecting Treponema pallidum in tissues. Furthermore, the ABC technique produced less intense nonspecific background staining than the IIP technique. Histologically, most of the treponemes were located in the upper dermis, epidermis and vessel walls in the order named, and rarely in the lower dermis of the syphilitic skin lesions.

Avidin↗
PubMed1994

Detection of Treponema pallidum by polymerase chain reaction in the cerebrospinal fluid of syphilis patients.

To find out if polymerase chain reaction could be used as a diagnostic tool for detecting neurosyphilis, we have applied the PCR for the detection of Treponema pallidum DNA in the cerebrospinal fluid (CSF) of syphilis patients. The results of the PCR of the CSF in 26 patients with at various stages of illness were compared with the results of other conventional tests used in the WHO criteria. T. pallidum was detected in the CSF of patients at all stages of syphilis, which indicates that they invade the central nervous system from the early stages of infection. However, the presence of T. pallidum in the CSF was not correlated with the results of other tests used in the WHO criteria, and its significance in the diagnosis of neurosyphilis should further be evaluated.

Cerebrospinal Fluid↗
PubMed2000

Virulent Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules and binding of T-lymphocytes to cultured human dermal microvascular endothelial cells.

Perivasculitis and endothelial cell abnormalities are prominent histopathologic features of syphilis. Various cutaneous lesions are the main clinical features of syphilis. We examined whether Treponema pallidum 47 kDa antigen regulates the expression of cell adhesion molecules on human dermal microvascular endothelial cells (HDMEC) and the regulation of T-lymphocytes binding to HDMEC. Using immunofluorescence flow cytometry and enzyme-linked immunosorbent assay (ELISA), we demonstrated that T. pallidum upregulated the expression of adhesion molecules, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. The 47 kDa antigen of T. pallidum also activated endothelium as measured by the upregulation of the expression of adhesion molecules on HDMEC, and it also promoted an increased adherence of T-lymphocytes to HDMEC. The expressions of ICAM-1 and VCAM-1 on HDMEC and the adherence of T-lymphocytes to HDMEC were inhibited by treatment with anti-TNF-alpha antibody or anti-IL-1alpha antibody. These results show that T. pallidum or T. pallidum-specific 47 kDa antigen are capable of stimulating HDMEC to increase the expression of ICAM-1, VCAM-1 and E-selectin and thereby, promote the adherence of T-lymphocytes. The whole process may play an important role in the immunopathogenesis of syphilis and it is likely that TNF-alpha and IL-1alpha are involved.

Antigens, Bacterial↗
PubMed2004

The effects of Treponema pallidum on human dendritic cells.

Cell mediated immune responses play a prominent role in syphilis, which is caused by Treponema pallidum. The role of dendritic cells (DC) in the syphilitic infection is not well understood in human. In the present study, we studied interaction of T. pallidum with DC, generated from human peripheral blood mononuclear cells with GM-CSF and IL-4. After adding T. pallidum for 16 hours to immature DC at culture day 7, the change of surface antigens on DC was monitored by flow cytometry, the amount of IL-12 in culture supernatant of DC was measured by ELISA and T cell stimulatory capacity of DC was checked in mixed lymphocyte reaction (MLR). We have observed an efficient phagocytosis of T. pallidum by electron microscopy as early as 2 hours after addition of T. pallidum to DC. Interaction of DC with T. pallidum resulted in increased surface expression of CD83 which was proportionally increased according to the number of T. pallidum. Expressions of CD80, CD86 and HLA-DR on DC were slightly increased. The amount of IL-12 in the culture supernatant of DC was increased (1,099 pg/ml) after the addition of T. pallidum. T. pallidum-infected DC also displayed enhanced T cell stimulatory capacity in MLR. As seen from the above, we observed phagocytosis of T. pallidum by DC as early as 2 hours after addition of T. pallidum to DC and found that T. pallidum can stimulate DC maturation which mean that DC modulate an protective immune response during T. pallidum infection.

Cells, Cultured↗
PubMed2001

The cutaneous response in humans to Treponema pallidum lipoprotein analogues involves cellular elements of both innate and adaptive immunity.

To extend prior studies implicating treponemal lipoproteins as major proinflammatory agonists of syphilitic infection, we examined the responses induced by intradermal injection of human subjects with synthetic lipoprotein analogues (lipopeptides) corresponding to the N termini of the 17- and 47-kDa lipoproteins of Treponema pallidum. Responses were assessed visually and by flow cytometric analysis of dermal leukocyte populations within fluids aspirated from suction blisters raised over the injection sites. Lipopeptides elicited dose-dependent increases in erythema/induration and cellular infiltrates. Compared with peripheral blood, blister fluids were highly enriched for monocytes/macrophages, cutaneous lymphocyte Ag-positive memory T cells, and dendritic cells. PB and blister fluids contained highly similar ratios of CD123(-)/CD11c(+) (DC1) and CD123(+)/CD11c(-) (DC2) dendritic cells. Staining for maturation/differentiation markers (CD83, CD1a) and costimulatory molecules (CD80/CD86) revealed that blister fluid DC1, but not DC2, cells were more developmentally advanced than their peripheral blood counterparts. Of particular relevance to the ability of syphilitic lesions to facilitate the transmission of M-tropic strains of HIV-1 was a marked enhancement of CCR5 positivity among mononuclear cells in the blister fluids. Treponemal lipopeptides have the capacity to induce an inflammatory milieu reminiscent of that found in early syphilis lesions. In contrast with in vitro studies, which have focused upon the ability of these agonists to stimulate isolated innate immune effector cells, in this study we show that in a complex tissue environment these molecules have the capacity to recruit cellular elements representing the adaptive as well as the innate arm of the cellular immune response.

Adolescent↗
PubMed2002

Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection.

Robust immune responses clear millions of treponemes to resolve lesions of primary and secondary syphilis, but cannot clear the treponemes that lead to debilitating and sometimes fatal tertiary syphilis. It is also known that the rabbit model and humans can be reinfected with heterologous isolates. How some treponemes are able to escape the immune system is unknown. In our laboratories rabbits immunized with the Seattle Nichols strain Treponema pallidum repeat protein K (TprK) were previously shown to have attenuated lesion development following challenge. In other isolates, TprK was shown to have seven discrete variable regions, with sequence variation among and within isolates. Using overlapping synthetic 20-aa peptides, we demonstrate that during experimental infection with the Nichols strain, the T cell responses are directed to conserved regions, while the Ab responses are directed primarily to variable regions. Abs from rabbits immunized with recombinant TprK recognized conserved and variable regions, suggesting that the conserved regions are inherently as immunogenic as the variable regions. TprK variability may allow some treponemes to escape recognition from Abs. The variable region heterogeneity may help explain the lack of protection against heterologous isolates.

Amino Acid Sequence↗
PubMedDate not supplied

Solid-phase hemadsorption assay for IgM antibody to Treponema pallidum in serum and umbilical cord blood.

A solid-phase hemadsorption assay (SPHA) for Treponema pallidum was evaluated using serum and umbilical cord blood. A total of 133 sera reactive using immunofluorescent antibody assay were tested by SPHA for IgM antibody against T. pallidum. They were categorized into clinical stages of syphilis, including eight sera with primary, 32 sera with secondary, 68 sera with latent, 21 sera with unclassified, and four sera with congenital syphilis. Sera were reactive for 75%, 90.6%, and 33.8% using SPHA in the primary, secondary, and latent phases, respectively. The method gave 57.1% reactivity for the unclassified phase of syphilitic sera and 100% reactive for the congenital syphilitic sera. All 80 immunofluorescent nonreactive sera were nonreactive by SPHA. Of 19 reactive cord blood samples tested, seven samples were reactive by SPHA. However, all 20 nonreactive umbilical cord blood samples were nonreactive by SPHA. The SPHA assay for the determination of IgM antibody to T. pallidum in serum appears to be sensitive for primary, secondary, and congenital syphilis. The SPHA assay in the cord blood is sensitive and specific.

Antibodies, Bacterial↗
PubMed1976

Swine dysentery: protection of pigs by oral and parenteral immunisation with attenuated Treponema hyodysenteriae.

An attenuated strain of Treponema hyodysenteriae was used to immunise 18 pigs in three experiments. Live attenuated spirochaetes were dosed orally and injected intra-peritoneally, and killed spirochaetes were injected intramuscularly with adjuvant. The vaccinated pigs, which developed high serum agglutination titres against T hyodysenteriae, and 18 unvaccinated litter-mates were repeatedly challenged with virulent T hyodysenteriae. Nine vaccinated pigs and 16 control pigs developed typical swine dysentery.

Agglutination Tests↗
PubMedDate not supplied

The microaerophilic nature of Treponema pallidum: enhanced survival and incorporation of tritiated adenine under microaerobic conditions in the presence or absence of reducing compounds.

Treponema pallidum, although sensitive to atmospheric concentrations of O2, requires low levels of O2 for optimal survival and metabolic activity. The addition of 0.0125-0.2 mg/ml of sodium metabisulfite to a basal medium consisting of Eagle's minimal essential medium and 50% fresh, heat-inactivated normal rabbit serum was found to have an effect similar to that of dithiothreitol in extending the survival of T. pallidum (Nichols strain) under 3% O2. Detailed analysis of the effect of O2 tension revealed that 50% motility was retained longest at atmospheric O2 concentrations of 1-5%, whether or not dithiothreitol or sodium metabisulfite were present. Concentrations of O2 of 3-10% were optimal for nucleic acid synthesis, as determined by [3H]adenine incorporation during the first 24 hr of incubation. Sodium metabisulfite was less effective than dithiothreitol in stimulating nucleic acid synthesis. Neither sodium metabisulfite nor dithiothreitol at their effective concentrations had any effect on levels of dissolved O2. During incubation under 3% O2, motility was maintained at > 50% for 15 days and virulence for at least 13 days by dilution of the treponemal suspensions every three days with fresh medium containing sodium metabisulfite. The optimal retention of motility and nucleic acid synthesis under microaerobic conditions in the absence of reducing compounds provides further evidence that T. pallidum is a microaerophilic organism.

Adenine↗
PubMed1998

Mitogenic activity of the outer membrane of Treponema denticola.

The outer membrane (OM) was isolated by detergent extraction from Treponema denticola ATCC 35405, ATCC 33521 and ATCC 35404, representing serovars a, b and c, respectively, as well as from two fresh isolates of T. denticola. Strict precautions were undertaken against the introduction of contaminant lipopolysaccharide when the OM was isolated. The OM was active in mitogenic stimulation of C3H/HeOuJ mouse spleen cultures, but to a somewhat lesser extent than purified lipopolysaccharide (LPS) from Escherichia coli 055:B5. Polymyxin B only partially inhibited the response. Unheated OM abrogated mitogenic activity of E. coli LPS, but heated preparations enhanced the mitogenic activity of E. coli LPS, suggesting the presence of a heat-labile cytolytic factor associated with T. denticola OM in addition to a putative lipopolysaccharide and/or heat-stable lipoprotein.

Animals↗
PubMed1999

Activation of human monocytic cells by Borrelia burgdorferi and Treponema pallidum is facilitated by CD14 and correlates with surface exposure of spirochetal lipoproteins.

Here we examined the involvement of CD14 in monocyte activation by motile Borrelia burgdorferi and Treponema pallidum. B. burgdorferi induced secretion of IL-8 by vitamin D3-matured THP-1 cells, which was inhibited by a CD14-specific mAb known to block cellular activation by LPS and the prototypic spirochetal lipoprotein, outer surface protein A. Enhanced responsiveness to B. burgdorferi also was observed when THP-1 cells were transfected with CD14. Because borreliae within the mammalian host and in vitro-cultivated organisms express different lipoproteins, experiments also were performed with "host-adapted" spirochetes grown within dialysis membrane chambers implanted into the peritoneal cavities of rabbits. Stimulation of THP-1 cells by host-adapted organisms was CD14 dependent and, interestingly, was actually greater than that observed with in vitro-cultivated organisms grown at either 34 degrees C or following temperature shift from 23 degrees C to 37 degrees C. Consistent with previous findings that transfection of Chinese hamster ovary cells with CD14 confers responsiveness to LPS but not to outer surface protein A, B. burgdorferi failed to stimulate CD14-transfected Chinese hamster ovary cells. T. pallidum also activated THP-1 cells in a CD14-dependent manner, although its stimulatory capacity was markedly less than that of B. burgdorferi. Moreover, cell activation by motile T. pallidum was considerably less than that induced by treponemal sonicates. Taken together, these findings support the notion that lipoproteins are the principle component of intact spirochetes responsible for monocyte activation, and they indicate that surface exposure of lipoproteins is an important determinant of a spirochetal pathogen's proinflammatory capacity.

Animals↗
PubMed2000

Clinical utility of a competitive ELISA to detect antibodies against Treponema pallidum.

Screening for Treponema pallidum infection is carried out on a large human population. To reduce costs, fewer tests which still offer adequate sensitivity and specificity could be performed. We studied the reliability of a novel indirect ELISA method to test for this infection. Several panels of sera were used that corresponded to 40 primary infections (group 1), 13 recurrences (group 2), 348 latent infections (group 3), 5 samples with anticardiolipin antibodies (group 4), 15 samples from patients with Lyme borreliosis (group 5), and 400 samples from blood donors and healthy pregnant women (group 6). The ELISA showed a global sensitivity and specificity of 100 and 99.5%, respectively. Our evaluation shows that Enzygnost Syphilis is a sensitive, specific, and simple test to screen for this infection.

Adult↗