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PubMed1996

A study of treponema pallidum haemagglutination positive blood donors in Singapore.

The Treponema Pallidum Haemagglutination (TPHA) test has been used for screening of syphilis among blood donors in Singapore since September 1992. Among the 79 500 donations that were screened between September 1992 and December 1993, 191 were tested positive (incidence rate of 0.24%). Seventy-two donors (37.7%) were evaluated at the Department of Sexually Transmitted Disease Control clinic. Seventy-nine percent of these donors had high risk sexual exposure and 33.3% had a history of sexually transmitted diseases. Eighty-three percent of the donors were diagnosed to have late latent syphilis, 9.7% were diagnosed to have a serological scar and the remaining had secondary syphilis, early latent syphilis and a false positive reaction. At least 65.3% of these donors would have been missed if the reagin test was used alone as the screening test. Thus, the TPHA test is a good marker for screening those who have high risk sexual behaviour and it is a more sensitive test than the reagin test for screening blood donors.

Adult↗

PubMed1997

In utero infection with Treponema pallidum in early pregnancy.

Amniocentesis was performed under sonographic guidance in gravidas (< 20 weeks' gestation) with untreated syphilis. Five to ten millilitres of amniotic fluid from each patient was used for rabbit infectivity testing (RIT) and polymerase chain reaction (PCR) to detect amniotic fluid infection with Treponema pallidum. Gravidas were treated with benzathine penicillin G. Newborns were examined for clinical and laboratory signs of congenital syphilis including immunoglobulin M (IgM) antibody to T. pallidum by Western blotting (immunoblotting). Eleven patients were enrolled at a mean gestational age of 16.8 weeks. T. pallidum was recovered from amniotic fluid by RIT in four cases (36 per cent), and PCR was positive in three of the amniotic fluid specimens (27 per cent). There were no false-positive PCR results. None of the newborns had clinical evidence of congenital syphilis and their sera lacked IgM reactivity to T. pallidum antigens by immunoblotting. These findings confirm in utero infection with T. pallidum in continuing early pregnancy and indicate that in utero treponemal infection can be eradicated by maternal treatment.

Adolescent↗

PubMed1982

Neurosyphilis: intrathecal synthesis of oligoclonal antibodies to Treponema pallidum.

Oligoclonal IgG was present in the cerebrospinal fluid (CSF) of each of three patients with neurosyphilis studied. Conventional serological tests disclosed reduced serum/CSF ratios of antibodies to Treponema pallidum (TP) in each patient, consistent with intrathecal production of treponemal antibodies. Antibody analyses of electrofocused specimens by an immunofixation technique disclosed intrathecal production of oligoclonal TP antibodies in all patients. Treponemal antibody light chains showed a close correlation with the light chains of the oligoclonal CSF IgG. Absorption of the CSF with TP caused removal of the oligoclonal IgG. The results provide strong evidence that the oligoclonal CSF IgG in neurosyphilis represents TP antibodies, reflecting a specific immune response in the central nervous system to the infectious agent.

Aged↗

PubMed1998

Illuminating the agent of syphilis: the Treponema pallidum genome project.

As the causative agent of the common sexually transmitted disease syphilis and a fastidious, microaerophilic obligate parasite of humans, Treponema pallidum subsp. pallidum is one of the few prominent infectious agents that has not been cultured continuously in vitro. T pallidum therefore represents an attractive candidate for genomic sequencing. Preliminary sequence results from the 1.13 million base pair genome are consistent with the expected limited metabolic capabilities of this spirochete, but indicate that the bacterium may express toxins and surface proteins that have not been identified previously.

Genome, Bacterial↗

PubMed1993

Phosphorylated nutrient uptake by Treponema denticola and other potential periodontopathogens.

Nutrient uptake may contribute to the survival of certain potential periodontopathogens in the competitive environment of the gingival sulcus. This study was conducted to assess the uptake of commercially available, key radiolabelled phosphorylated compounds by Treponema denticola. There was a linear relationship in the uptake of ATP and G-1-P with cell concentrations of 0.1-2.5 mg of cell dry weight. This uptake reached a steady state within 15 min, and at temperatures of 25-37 degrees C the uptake ranged between 13-21%. At 50 degrees C, or above 50 degrees C the assay cells took up 0-1% of the assay nutrients. The maximum uptake of ATP was 47.5, 38 and 47.6 pmoles for serovars a, b and c. G-1-P yielded maximum uptake values of 306, 304, and 306 pmoles respectively. Significant uptake of ATP, or G-1-P occurred at pH 4.8 for serovars a, or c, while serovar b showed a broad pH range for the uptake of G-1-P. Stannous fluoride at a concentration of 0.17 mM enhanced the uptake of ATP while chlorhexidine digluconate inhibited the uptake of ATP. At concentrations usually employed (0.05-0.5 mM) the metabolic poisons 2,4-dinitrophenol, sodium azide, sodium arsenite and the ATPase inhibitor N,N1-dicyclohexylcarbodiimide did not alter significantly the uptake of ATP. The uptake of ATP, by Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Fusobacterium nucleatum and T. denticola at pH 4.8 was 13.4, 1.5, 6.9, and 18-20.4%, respectively. At pH 6.5 the uptake for the microorganisms indicated above was 22, 1.3, 6.6 and 6.2%, respectively. G-1-P showed the same trend in its uptake by A. actinomycetemcomitans, P. gingivalis, F. nucleatum and T. denticola. This study provides for the first time specific examples of phosphorylated nutrient uptake by potential periodontopathogens, and that the uptake of phosphorylated nutrients can be suppressed by oral hygienic compounds.

Adenosine Triphosphate↗

PubMed1995

Purification and general properties of an oligopeptidase from Treponema denticola ATCC 35405--a human oral spirochete.

An endo-acting oligopeptidase (OPase) was purified to homogeneity from the cells of Treponema denticola ATCC 35405--a human oral spirochete--by a procedure that comprised a mild Triton X-100 extraction (which disintegrates the outer membrane but leaves the cells morphologically intact) and four successive fast protein liquid chromatographic steps of the extract. The activity of this oligopeptidase (formerly named "trypsin-like" enzyme and "BANA-peptidase") together with the proteinase activities of T. denticola and Porphyromonas gingivalis is utilized in a diagnostic test for human periodontal infections, but the enzyme's chemical nature has not been studied. The enzyme is a cell-associated 78-kDa protein with an isoelectric point of 6.1, and its estimated minimum peptide length was 688 amino acid residues. The OPase does not hydrolyze proteins, but hydrolyzes -X-Arg-p-nitroaniline peptides between arginine and the chromogen, the optimum pH of hydrolysis covering a broad pH range (7 to 9). The OPase is not a metalloenzyme, although 1.0 mmol/liter Ca(II) increases the rate of the hydrolysis of all substrates. Ca(II) did not affect the values of the Michaelis constant. The OPase activity is not dependent on reactive SH-groups, but is suggested to depend on the catalytic triad COOH. . .His. . .Ser. The N-terminal sequence for the first 29 amino acid residues is MKQSDFEKPPIAEIKETRFEKFGKTRIDN. The purified enzyme is very sensitive to chlorhexidine acetate (mixed inhibition; Ki = 0.85 microM) and somewhat less sensitive to bacitracin (Ki(app) = 27.5 microM). The present OPase is considered to belong to the serine peptidases, functionally resembling trypsin except that the OPase does not hydrolyze proteins. The OPase may be regarded as an oligopeptidase, the substrate specificity profile of which resembles to a certain extent that of some members of the coagulation cascade.

Amino Acid Sequence↗

PubMed1999

The time-dependent clearance of virulent Treponema pallidum in susceptible and resistant strains of guinea pigs is significantly different.

The kinetics of clearance of Treponema pallidum spp. pallidum Nichols from skin and testes of susceptible C4-deficient (C4D) and -resistant Albany (Alb) strains of guinea pigs (gps) was evaluated using the polymerase chain reaction (PCR) and the rabbit infectivity test (RIT). For each strain there were two groups of animals, one infected with virulent T. pallidum (TP) and one control injected with heat-killed treponemes (HKTP). The kinetic studies and their statistical analysis showed that in the C4D strain the microbial clearance in both tissues was significantly slower (p < 0.005) and still incomplete at 3 months after infection. In the Alb strain the clearance was faster and apparently completed within a month. A greater permissiveness in bacterial growth in C4D compared to Alb appears to be one critical factor determining the different rate of local elimination after primary infection. In both strains there was some correlation between the severity and duration of cutaneous lesions and the local persistence of viable organisms. This correlation was not observed in testes. These studies suggest a genetic basis for the strain-specific susceptibility and resistance phenotypes in the pathogenesis of syphilis.

Animals↗

PubMed1993

Cytotoxic effect of peptidoglycan from Treponema denticola.

Peptidoglycan isolated from pathogenic bacteria has been previously found to exhibit various biological activities. The aim of this investigation was to evaluate the toxicity of Treponema denticola peptidoglycan towards epithelial cells. The cytotoxicity of a lipopolysaccharide-like material was also determined. Epithelial cells were incubated with the test substances and cell viability was assayed by measuring lactate dehydrogenase activity and the conversion of tetrazolium salt into blue formazan. Morphological changes in the epithelial cells were monitored by scanning and transmission electron microscopy. While lipopolysaccharide-like material exerted negligible toxic effects on the epithelial cells, peptidoglycan was highly toxic. This cytotoxicity was both time- and concentration-dependent and was higher in the presence of serum. The epithelial cells appeared to be unable to recover following a short period of incubation with peptidoglycan. The study demonstrated that peptidoglycan from T. denticola is a potent toxic factor for epithelial cells. This represents a potential new virulence factor for this suspected periodontopathogen.

Animals↗

PubMed1994

Characterization of hemolysis and hemoxidation activities by Treponema denticola.

This study determined the presence of two hemolytic activities in the oral treponeme, Treponema denticola, strains ATCC 35404 (TD-4), ATCC 33520, GM-1, and MS25. These activities, referred to as hemolytic and hemoxidative (HeA, HeO, respectively), were found to be both secreted into the extracellular environment, and cell associated. The extracellular activity was associated with small molecules with relative molecular weights of < 1000 Da, and its activity was cysteine independent; the cell-associated HeA and HeO activities were associated with a molecular weight fraction > 10 kDa, and were cysteine dependent. The HeO activity of the fractionated material observed was due to the oxidation of hemoglobin to methemoglobin, and preceded the HeA lysis of the RBCs by approximately 2 h. Heating at 80 degrees C and treatment with proteinase K resulted in the complete destruction of these activities in the fraction > 10 kDa, while lipase at high concentration (800 micrograms/ml) reduced the HeA and HeO activities in the extracellular fraction by approximately 50%. Proteinase inhibitors had a variable effect on HeA and HeO activities in both extracellular and cell-associated fractions. Scanning and transmission electron microscopy revealed a progressive destruction of the RBC membrane, with membrane protrusions formed early in the interaction, which progressed to irregular holes in the membrane, and the complete loss of membrane integrity.

Erythrocytes↗

PubMed1994

Purification and characterization of a 45 kDa hemolysin from Treponema denticola ATCC 35404.

A 45 kDa polypeptide capable of erythrocyte (RBC) lysis and hemoglobin oxidation was isolated from Treponema denticola, ATCC 35404 (TD-4) after sequential ammonium sulfate (2.8-3.6 M) precipitation and preparative electrophoresis. The purified polypeptide produced a single protein band on PAGE at a relative molecular weight of 45 kDa in the presence and absence of SDS. The polypeptide was sensitive to proteinase K and pronase, and heating at 80 degrees C. The protease inhibitors, PMSF, TLCK and benzamidine had no inhibitory affect on activity. It was non heat-modifiable, and lost all hemolytic and hemoxidative function in SDS. Cysteine and other sulfhydryl-containing compounds were required for hemolytic and hemoxidative activities. The isoelectric point of the polypeptide was 5.3 and N'-terminal sequence analysis indicated it to belong to a new, so far undescribed group of peptides possessing hemoxidation and hemolytic activities. Functionally, it was capable of rapid hemoxidation of sheep and human erythrocytes (hemoglobin to methemoglobin) coupled to erythrocyte lysis, or hemolysis.

Amino Acid Sequence↗

PubMed1994

Effect of iron regulation on expression and hemin-binding function of outer-sheath proteins from Treponema denticola.

The effect of the iron-chelating compounds EDDA and BPD on polypeptide regulation in the putative oral pathogen Treponema denticola was studied. SDS-PAGE analysis of the T. denticola strains grown in the presence of EDDA or BPD, i.e. iron-limiting environmental conditions, revealed the expression of 44 and 43 kDa polypeptides in the outer sheath, a 73 kDa polypeptide in the cell membrane, and a 16 kDa polypeptide in the soluble cell fraction. The hemin-binding activity of purified outer sheaths from T. denticola TD-4 grown in the presence of 6.4 mM EDDA was significantly greater than that observed in control (absence of EDDA) outer sheaths. Both activities were inhibited by proteinase K. SDS-PAGE, LDS-PAGE and TMBZ staining revealed the 44 and 43 kDa outer-sheath polypeptides to be expressed by T. denticola strains GM-1. MS-25, ATCC 33520 and ATCC 33404 (TD-4), strains which possessed strong hemin-binding activity. The 44 kDa hemin-binding polypeptide was purified by 1% CHAPS solubilization, HPLC, and SDS-preparative electrophoresis. N'-terminal sequence analysis indicated the purified 44 kDa polypeptide to belong to a new, undescribed group of polypeptides possessing hemin-binding activity.

2,2'-Dipyridyl↗

PubMed1994

Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli.

The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis. In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete. Our laboratory has been investigating the pathobiology of T. denticola, and has demonstrated the production of several hemolysins by T. denticola. In this report two hemolysin genes from T. denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T. denticola on sheep blood agar plates. Physical maps of the insert fragments were not identical. Southern blot analyses suggested some degree of homology in the nucleotide sequence. Maxicell analyses of [35S]-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide. Biochemical characterization of the T. denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA. Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids. Iron partially inhibited the hemolytic activities. Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation. These results suggest the isolation of an identical hemolysin gene from T. denticola strains TD-4 and GM-1.

Animals↗

PubMed1989

Interactions of Treponema pallidum with endothelial cell monolayers.

Syphilis is a chronic disease characterized by hematogenous dissemination of Treponema pallidum into tissues such as the cardiovascular and central nervous systems. In order to test whether these aspects of the pathogenesis of syphilis reflect an ability of T. pallidum to invade vascular endothelial surfaces, we explored the association of T. pallidum with human and rabbit endothelial cells in vitro. Using radiolabeled motile organisms, we found that treponemal attachment was two times greater to rabbit aortic endothelial cells and human umbilical endothelial cells than to HeLa cells. Mild trypsinization of attached treponemes resulted in release from cells of all organisms detectable by darkfield microscopy without visible damage to the monolayer. Nevertheless, 25% of the counts representing T. pallidum remained associated with the cell monolayers. Further trypsin treatment to release the monolayer and differential centrifugation showed that 80% of the remaining cell-associated counts were not within the cells. These results suggest that some treponemes had associated with the monolayer in a trypsin resistant niche. Additionally, motile T. pallidum passed through tight-junctioned endothelial cell monolayers on membrane filters under conditions were heat-killed T. pallidum and the host indigenous nonpathogen. T. phagedenis biotype Reiter failed to do so. Electron micrographs of transverse sections through the monolayers showed many T. pallidum in junctions between endothelial cells. These studies suggest that T. pallidum may leave the circulation by passing between endothelial cells.

Animals↗

PubMed1993

Development of quasi-multicellular bodies of Treponema denticola.

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes,forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.

Freeze Fracturing↗

PubMed1983

Restriction analysis of DNA from Treponema pallidum, the causative agent of syphilis.

When purified DNA from pathogenic Treponema pallidum is digested with restriction endonucleases it results in the formation of discrete DNA fragments which range between 2.5 to 10 Kilobase pairs. No such precise fragmentation occurs with DNA isolated from nonpathogenic T. pallidum. The appearance of the discrete restriction fragments from the pathogenic T. pallidum DNA does not represent a contamination of satellite DNA from rabbit, the host in which the organism was propagated, but rather represents the presence of redundant DNA or DNA of less complexity in the pathogenic T. pallidum DNA preparation.

Centrifugation, Density Gradient↗

PubMed1990

Growth stimulation of Treponema denticola by periodontal microorganisms.

Previous experiments have indicated that enrichment of subgingival plaque in human serum can lead to the accumulation of Treponema denticola. T. denticola depends on bacterial interactions for its growth in serum. Aim of the present study was to identify specific microorganisms involved in the growth stimulation of T. denticola. To this end, strains isolated from previous plaque enrichment cultures were tested for growth stimulation in co-cultures with T. denticola. In addition, growth of T. denticola was tested in culture filtrates of the same strains, Bacteroides intermedius, Eubacterium nodatum, Veillonella parvula and Fusobacterium nucleatum were found to enhance growth of T. denticola in co-cultures. A continuous co-culture of T. denticola, F. nucleatum and B. intermedius in human serum gave very high levels of T. denticola, up to 3.10(9).ml-1. Mechanisms involved in growth stimulation may include the ability of B. intermedius and E. nodatum to cleave the protein-core of serum (glyco-)proteins, making these molecules accessible for degradation by T. denticola. In addition, E. nodatum was found to produce a low-molecular weight growth-factor for T. denticola, that was heat-stable and acid as well as alkaline resistant. V. parvula may provide peptidase activities complementary to those of T. denticola. The nature of the growth enhancing activity of F. nucleatum is yet unknown. The data support the dependency of T. denticola on other bacterial species for growth in the periodontal pocket.

Amino Acid Sequence↗

PubMed1979

Characterization of the cytoplasmic fibrils of Treponema refringens (Nichols).

The cytoplasmic fibrils of Treponema refringens were studied in situ by electron microscopy of thin sectioned and negatively stained cells. From 5 to 21 parallel fibrils ran through the cell in a band adjacent to the inner side of the cytoplasmic membrane, on the inner sides of the curves of the spirochete. The nuclear areas of cells were adjacent to the fibrils. Cross sections of fibrils isolated from cells which had been lysed were polygonal and not uniformly electron dense. Polyacrylamide gel electrophoresis of partially purified fibril preparations indicated their main component to be a protein with a molecular weight of 97,000. Fibrils were solubilized by 1% trypsin, 1% pronase, 6 M urea, 1 N HCl, 0.005 N NaOH or 1.3% sodium dodecyl sulfate. By electron microscopy of negatively stained isolated fibrils, each fibril was found to be a complex arrangement of strands rather than a single tubule.

Cell Fractionation↗

PubMed1978

Morphology of freeze-etched Treponema refringens (Nichols).

The freeze-etch technique was used to study the morphology of Treponema refringens (Nichols). There is a single band of cytoplasmic fibrils which follows a path in the form of a right-handed helix with a periodicity of 1500 nm around the body of the treponeme just below the cytoplasmic membrane. There are two major fracture planes, one located in the interior of the outer envelope and the second in the interior of the cytoplasmic membrane. The "blebs" or "surface protuberances", which are quite prominent in negative-stained preparations, were not evident with freeze-etch preparation, indicating they are not a part of the normal structure of this organism. The outer envelope in untreated cells was observed to closely fit the body of the treponeme, whereas the outer envelope of glutaraldehyde-treated cells had a loose, wrinkled appearance. Thus the "loose-fitting" outer envelope generally described for treponemes is most likely an artifact of preparation for negative-staining and thin-sectioning.

Cell Membrane↗