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PubMed1993

Reactivity of locally produced CSF antibodies in patients with neurosyphilis against antigens of Treponema pallidum.

The reactivity and specificity of locally produced cerebrospinal fluid (CSF) antibodies against antigens of Treponema pallidum were assessed by Western blotting in patients with clinical signs of parenchymal or meningovascular neurosyphilis. All nine patients showed local production of treponeme-specific antibodies in the central nervous system (CNS). In most of the patients serum and CSF antibodies were bound to the same antigens: the common treponemal 48/45 kDa protein and the putative specific T. pallidum protein in the range of 12-14 kDa. In some patients the intensity of staining obtained by CSF antibodies was higher than that derived from serum, indicating locally produced antibodies. In contrast to other more acute inflammatory CNS diseases, no expanded or different antigen binding of the CSF antibodies compared with serum antibodies was found in neurosyphilic patients. The results presented are discussed with regard to the role of the blood-brain barrier in antibody concentrations of CSF and serum.

Adult↗
PubMed1988

Ig class and IgG subclass responses to Treponema pallidum in patients with syphilis.

The Ig class and IgG subclasses of anti-Treponema pallidum antibodies in human serum were quantified using solid-phase enzyme-linked immunosorbent assays. Development of these assays with monoclonal antibodies, each specific for a human immunoglobulin class or IgG subclass, provided quantitative data concerning the major antibody specificities. In patients with primary syphilis, anti-T. pallidum activity was limited almost exclusively to IgG1 and IgM. Coordinate, restricted expression of IgG1 and IgG3 responses in T. pallidum-specific assays was observed with sera from patients with active secondary syphilis. IgG1 and IgG3 accounted for roughly 53 and 43% of the total anti-treponemal IgG antibody activity, respectively. While IgM antibody levels were elevated in the patients with secondary syphilis, IgG2 and IgG4 levels, if present at all, represented less than 10 and 2% of the total IgG activity, respectively. Ig in sera from patients who had been treated adequately for secondary syphilis were restricted almost entirely to IgG3 and IgG1. Considering the low level of IgG3 in serum, disproportionately high percentages of antitreponemal antibodies were found in this subclass during and after treatment for secondary syphilis. The restricted, coexpression of the IgG1 and IgG3 isotypes may reflect the close genetic linkage of the gamma 1 and gamma 3 genes and possibly the impact of immunoregulatory mechanisms in response to the induction and expression of autoantibodies which arise during the course of secondary syphilis.

Adolescent↗
PubMed1984

Low molecular weight-IgM antibody in syphilis detected by Treponema pallidum immune adherence (TPIA) test.

Sera obtained from patients with syphilis were subjected to gel filtration chromatography to estimate antibody activity in the fractions by means of Treponema palludum immune adherence (TPIA) test. Antibody activity was found in IgM and IgG fractions and in the intermediate fraction between them. The TPIA activity of the intermediate fraction was depleted by an immunosorbent coupled with anti-mu chain antibody but not by that coupled with anti-alpha chain antibody. After separation by gel filtration and affinity chromatography, the fraction with the TPIA activity showed a positive precipitation reaction against anti-mu chain antiserum but a negative reaction with anti-alpha chain or anti-gamma chain antiserum. The antibody activity in this fraction was thus confirmed to be mainly due to low molecular weight-IgM antibody. In untreated patients with primary and secondary syphilis, the TPIA activity was demonstrated with high frequency in the low molecular weight-IgM fraction and it decreased gradually after treatment, although lowered activity persisted over a long period of time. Late syphilis cases did not show any appreciable change in TPIA activity in this fraction even after treatment.

Antibodies, Bacterial↗
PubMed1984

[1st results with the Treponema pallidum hemagglutination (TPHA) test in blood stains].

Bloodstains were produced from probationers who had had syphilis at some time in their lives and from others whose anamnesis had no indication of syphilis. After storage the stain eluates underwent the treponema-pallidum-haemagglutination (TPHA) test, with the eluates' IgG content being adapted to a concentration adequate to the test conditions. The results received from the stain eluates of the previous syphilis patients corresponded in 85% of the cases with the serum findings of these probationers. It appears that the TPHA test can already provide clues as to the identity of an unknown stain producer at the beginning of the police investigation. Methodical parallels to dried-blood tests of syphilis as a clinical problem will be discussed.

Adult↗
PubMed1981

Role of serum in survival of Treponema pallidum in tissue culture.

During attempts to cultivate Treponema pallidum, it was determined that length of time for survival of virulent treponemes was highly dependent on the quality of the fetal bovine serum (FBS) used as a protein supplement in the culture medium. Eighteen lots of commercial FBS were tested for their ability to maintain survival of T. pallidum in cultures of cottontail rabbit epithelial (SflEp) cells. All were capable of supporting growth of these cells. However, in tests on five of the lots, attachment of treponemes to the SflEp cells was either extremely poor or the 50% survival time (ST50) was less than 5 days. With two of these lots, no treponemes survived for 5 days. By contrast, in tests with 11 of the FBS lots, the ST50 of the treponemes was 12 days or greater; however, there was a great variation in the number of treponemes that attached. Selection of lots of FBS for ultimate experimental use was based on their influence both to extend length of time for survival of treponemes and to increase the number of treponemes that attached to the SflEp cells during that period.

Animals↗
PubMed2001

Preliminary characterization of a tentatively novel rumen bacterial species from the genus Treponema.

A new spirochetal strain was isolated from the rumen of a black-and-white Holstein cow and preliminarily characterized. The sugar fermentation tests and morphological observations indicated this organism to be a member of a novel, as yet undescribed spirochetal rumen species. The small subunit ribosomal RNA genes were amplified and the PCR products were cut with the restriction endonucleases TaqI, DdeI, HhaI and Sau3AI. The comparison of the observed RFLP with the hypothetical fragment lengths of the computer analyzed 16S rRNA sequences from the type strains of the ruminal spirochetes Treponema bryantii and T. saccharophilum confirmed the tentative novel identification. Transmission electron microscopy showed that the bacterium has the typical spirochetal structures, i.e. the outer sheath, the protoplasmic cylinder and the axial filament (it is not yet clear how many flagella compose the filament). An additional extracellular structure was observed which appeared as an exocytoplasmic polar flagellum, approximately 2 microns long and protruding from one tip of the cell. The average size of the cells was 0.5 x 10-15 microns and the wavelengths and the amplitudes of the primary coils were 2.9 and 1.3 microns, respectively.

Animals↗
PubMed2001

Selenium-dependent growth of Treponema denticola: evidence for a clostridial-type glycine reductase.

Assessment of the nutritional requirements of Treponema denticola disclosed a strict growth dependence on selenium. In vivo labeling of cells of this organism with (75)Se and electrophoretic analysis revealed three labeled bands, two of which were selenoproteins correlating in size with subunits A and B of glycine reductase. Antibodies directed against glycine- or betaine-reductase subunits of Eubacterium acidaminophilum specifically also reacted with proteins from cell lysates of T. denticola. Moreover, ORFs within the T. denticola genome sequence were found whose products display high sequence similarity to glycine-reductase subunits. These findings strongly support the notion that T. denticola ferments amino acids via the activity of glycine reductase, an enzyme previously thought to be restricted to gram-positive bacteria.

Amino Acid Oxidoreductases↗
PubMed2001

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene.

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.

Aerobiosis↗
PubMed2004

Expression of Treponema denticola oligopeptidase B in Escherichia coli.

Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases. This bacterium possesses a particular arginine peptidase activity (previously called "BANA-peptidase" or "trypsin-like enzyme") that is common to the three cultivable bacterial species most highly associated with severe periodontal disease. We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T. denticola through DNA sequencing and mutagenesis studies. In the present study, we report expression of T. denticola OpdB peptidase in Escherichia coli. The opdB PCR product was cloned into pET30b and then transformed into the E. coli BL21 (DE3)/pLysS expression strain. Assays of enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola. Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.

Bacterial Proteins↗
PubMed2003

Phagocytosis of Treponema pallidum and reactive oxygen species production by isolated rat Kupffer cells.

The in vitro phagocytosis of viable Treponema pallidum subsp. pallidum by isolated rat Kupffer cells, studied by immunofluorescence staining of Kupffer cells-associated bacteria, showed that ingestion of live, unopsonized treponemes was slow: in fact, Kupffer cells started to be positive 1 h after infection, when only 4% of the cells presented small round fluorescent inclusion-like bodies. Thereafter, the number of positive cells progressively increased with time: 7%, 17%, 36%, and 69% of Kupffer cells were positive, respectively, 2, 4, 6 and 8 h after infection. Opsonization of T. pallidum with human immune serum did not substantially modify the percentage (8%) of Kupffer cells ingesting T. pallidum 1 h after infection, whereas opsonization significantly ( P<0.01) increased phagocytosis after 2, 4 and 6 h of incubation, when 44%, 58%, and 68% of Kupffer cells were positive, respectively. At 8 h after infection of Kupffer cells by opsonized T. pallidum, 75% of the cells were positive by immunofluorescence. Heat-inactivation of T. pallidum slightly enhanced phagocytosis. In contrast, opsonization of heat-inactivated spirochetes with specific antibodies significantly ( P<0.01) increased the phagocytosis of bacteria by Kupffer cells, beginning as early as 30 min after infection, when 65% of the cells were positive by immunofluorescence. The reactive oxygen species (ROS) production by Kupffer cells following incubation with spirochetes was also determined by chemiluminescence. Treponemes induced an oxidative burst in Kupffer cells in a dose-dependent manner and the generation of ROS was already detectable 20 min after the exposure of the Kupffer cells to treponemes and peaked at 35 min of incubation. Live, as well as live and opsonized, and heat-inactivated treponemes, induced an O(2)(-) production lower than that induced by heat-inactivated and opsonized spirochetes.

Animals↗
PubMed2004

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin.

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

Bacterial Proteins↗
PubMed2006

The first crystal structure of class III superoxide reductase from Treponema pallidum.

Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 A resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the beta-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.

Amino Acid Sequence↗
PubMed1995

Induction of cytoskeletal rearrangements and loss of volume regulation in epithelial cells by Treponema denticola.

The early responses of oral epithelial cells to the adhesion of the oral spirochaete Treponema denticola were studied as a model of microbial perturbation of the plasma membrane. KB cell (ATCC CCL 17) monolayers were incubated with T. denticola (ATCC 35405) in alpha-MEM (minimal essential medium) for periods of 1-4 h at 37 degrees C without serum. Control cultures were exposed to bacteria-conditioned alpha-MEM without serum or bacteria or to alpha-MEM alone. At the end of each incubation, detached and attached epithelial cells were harvested and analysed separately. Compared with controls, T. denticola induced in 25% of cells a two-fold, time-dependent increase of detachment by 4 h. Detached cells in both T. denticola-exposed and control cultures exhibited 25% reductions in modal diameter, did not exclude propidium iodide, did not readhere, and did not form colonies. In T. denticola-exposed cultures, a larger subset (75%) of cells remained attached to the substratum, demonstrated no significant reduction of colony-forming efficiency and excluded propidium iodide. However, these cells exhibited a 21% reduction in diameter (p < 0.05), a 60% decrease of F-actin (p < 0.001), and a 74% reduction in the proportion expressing desmoplakin II (p < 0.01) after exposure to T. denticola. Flow cytometry showed a small (14%) but significant (p < 0.001) reduction in mean fluorescence intensity due to keratin expression in T. denticola-treated cultures. Exposure of cells to anisosmotic media demonstrated that, in contrast to controls, cultures challenged by bacteria failed to undergo compensatory volume regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1986

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans.

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.

Animals↗
PubMed1985

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

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

Adhesins, Bacterial↗
PubMed1996

Detection of antigenic determinants in the Treponema pallidum membrane protein TmpA using overlapping synthetic peptides.

The antigenic structure of the 42 kDa membrane protein of Treponema Pallidum, TmpA, was studied using synthetic peptides. Ten overlapping peptides, 35-40 residues each, were synthesized in order to cover the entire sequence of the molecule. The antigenic activity of the fragments was examined by enzyme-linked immunosorbent assay (ELISA). In this way it was possible to demonstrate a significant antigenic activity of four peptides which were reactive with syphilitic sera. The N-terminal fragment TmpA1, 38 residues long, proved to be the most reactive. Its antigenic structure was therefore studied in more detail, by examining shorter fragments. The N-terminal portion of TmpA1, consisting of 19 residues, (ASGAKEEAEKKAAEQRALL) represents an important fragment of the molecule, and was specifically interactive with most of the syphilitic sera examined.

Amino Acid Sequence↗