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PubMed1981

Cultivation of pathogenic Treponema pallidum in vitro.

Treponema pallidum was discovered relatively late and was not cultured in vitro. Both the delineation of T. pallidum biology and the eradication of syphilis suggest the necessity of cultivation in vitro. An attempt has been made with an improved medium to cultivate pathogenic T. pallidum Budapest strain in vitro. Only in the first passage, evidence of in vitro multiplication of T. pallidum has been established by (i) macroscopic observation, (ii) darkfield examination, (iii) electron microscopic examination, (iv) optical densities, (v) tritium labelled thymidine incorporation, and (vi) the pathogenicity off the cultured organisms was evidenced by rabbit challenge. Explanation of the oxygen utilization of T. pallidum suspension is discussed. Unidentified formations were observed on electron micrographs from the 96 h cultures. They may belong to the multiplication forms of treponemes. Further experiments are needed for their identification and for expansion of the multiplication of T. pallidum beyond the first passage.

Animals↗
PubMed1978

Effects of fatty acids on motility retention by Treponema pallidum in vitro.

Treponema pallidum (Nichols virulent strain) was incubated under 75% N2 + 20% H2 + 5% CO2 in prereduced serum-free modified Eagle-Richter medium supplemented with different concentrations of various long-chain fatty acids complexed with fatty acid-free bovine serum albumin. Motility retention was greater in medium with oleic acid containing 15 rather than 2 mg of albumin per ml. Palmitic, stearic, oleic, or linoleic acid alone caused rapid loss of motility at concentrations as low as 5 microgram/ml. Elaidic acid (92 microgram/ml) alone had no effect on motility. Various combinations of saturated plus unsaturated fatty acids did not inhibit motility retention or were less inhibitory than either of the individual fatty acid components. The combination of palmitic plus oleic acids was least toxic. Rapid loss of motility occurred with pairs of unsaturated or saturated fatty acids, or with Tween 40, 60, or 80, alone or combined. Autoxidation of oleic acid resulted in decreased toxicity for T. pallidum but increased toxicity for baby hamster kidney cells.

Culture Media↗
PubMed1980

Long-term incorporation of tritiated adenine into deoxyribonucleic acid and ribonucleic acid by Treponema pallidum (Nichols strain).

Treponema pallidum (Nichols strain), extracted in medium containing Eagle minimal essential medium 50% fresh, heat-inactivated normal rabbit serum, and 1.0 mM dithiothreitol, was incubated under 3% oxygen in the presence of tritiated nucleic acid precursors. [8-3H]adenine was incorporated with high efficiency into trichloroacetic acid-insoluble material; 2'-deoxyadenosine and uridine were incorporated in lower quantities, and thymine and thymidine were not incorporated. Incorporation of [3H]adenine was inhibited by penicillin G, mitomycin C, actinomycin D, and erythromycin, but was not affected by cycloheximide. Partial purification of nucleic acids from T. pallidum incubated with [8-3H]adenine for 36 to 72 h and subsequent treatment with ribonuclease and deoxyribonuclease revealed that 15 to 20% of the trichloroacetic acid-precipitable counts were resistant to ribonuclease but susceptible to deoxyribonuclease. A simple assay was developed in which NaOH treatment was used to distinguish incorporation into ribonucleic acid and deoxyribonucleic acid. Both ribonucleic acid and deoxyribonucleic acid synthesis continued for 6 days of incubation under 3% O2, whereas incorporation was limited to the first day of incubation in samples incubated under aerobic or anaerobic conditions. T. pallidum thus appears to be capable of significant de novo deoxyribonucleic acid and ribonucleic acid synthesis under microaerobic conditions.

Adenine↗
PubMed1995

Pathology of the umbilical cord in congenital syphilis: analysis of 25 specimens using histochemistry and immunofluorescent antibody to Treponema pallidum.

Identification of Treponema pallidum in the placenta is important for diagnosis of congenital syphilis; however, spirochetes are difficult to observe in chorionic villi. To determine the sensitivity of umbilical cord examination for T pallidum, and the association of spirochetes with cord pathology, placentas were prospectively obtained from 25 women with untreated syphilis. The most common finding using hematoxylin-eosin staining was a normal-appearing umbilical cord (48%); necrotizing funisitis was the most frequent pathological lesion (36%). Spirochetes were detected using silver and immunofluorescent staining in 89% of cords, including 92% of histologically normal and 84% of abnormal cords. Three specimens showed subamnionic aggregates of spirochetes, consistent with amniotic fluid infection. Necrotizing funisitis was strongly associated with umbilical artery infection by spirochetes (P = .008). There was a 100% correlation between results of silver and immunofluorescent staining. The umbilical cord is a sensitive site for morphological confirmation of T pallidum; it is significant for the pathologist that spirochetes may often be detected in the absence of overt tissue inflammation or necrosis.

Antibodies, Bacterial↗
PubMed1997

Tromp1, a putative rare outer membrane protein, is anchored by an uncleaved signal sequence to the Treponema pallidum cytoplasmic membrane.

Treponema pallidum rare outer membrane protein 1 (Tromp1) has extensive sequence homology with substrate-binding proteins of ATP-binding cassette transporters. Because such proteins typically are periplasmic or cytoplasmic membrane associated, experiments were conducted to clarify Tromp1's physicochemical properties and cellular location in T. pallidum. Comparison of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities of (i) native Tromp1 and Tromp1 synthesized by coupled in vitro transcription-translation and (ii) native Tromp1 and recombinant Tromp1 lacking the N-terminal signal sequence revealed that the native protein is not processed. Other studies demonstrated that recombinant Tromp1 lacks three basic porin-like properties: (i) the ability to form aqueous channels in liposomes which permit the influx of small hydrophilic solutes, (ii) an extensive beta-sheet secondary structure, and (iii) amphiphilicity. Subsurface localization of native Tromp1 was demonstrated by immunofluorescence analysis of treponemes encapsulated in gel microdroplets, while opsonization assays failed to detect surface-exposed Tromp1. Incubation of motile treponemes with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazarine, a photoactivatable, lipophilic probe, also did not result in the detection of Tromp1 within the outer membranes of intact treponemes but, instead, resulted in the labeling of a basic 30.5-kDa presumptive outer membrane protein. Finally, analysis of fractionated treponemes revealed that native Tromp1 is associated predominantly with cell cylinders. These findings comprise a body of evidence that Tromp1 actually is anchored by an uncleaved signal sequence to the periplasmic face of the T. pallidum cytoplasmic membrane, where it likely subserves a transport-related function.

Azirines↗
PubMed1982

Effects of anaerobic and microaerophilic conditions of extraction and incubation on the survival of Treponema pallidum in vitro.

Treponema pallidum extracted from infected rabbit testes under anaerobic conditions survived longer in vitro than those extracted under aerobic conditions. Anaerobically extracted treponemes were incubated anaerobically for 0, 12, 24, 36, or 48 hours and then exposed to microaerophilic conditions (3% oxygen) for further incubation. Treponemes transferred to microaerophilic conditions after 36 or 48 hours' anaerobic incubation maintained significantly greater viability compared with those kept under constant microaerophilic conditions, although there was no difference after 12 or 24 hours. T pallidum incubated under constant anaerobic conditions, however, usually maintained greater viability than those kept under constant microaerophilic conditions. These results suggest that T pallidum is sensitive to oxygen toxicity both during initial extraction from orchitic rabbit testes and subsequent incubation in vitro. In the latter case, it can be partially protected by a period of anaerobic incubation in vitro, before exposure to microaerophilic conditions.

Anaerobiosis↗
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↗
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↗
PubMed1988

[In vivo study of the sensitivity of Treponema pallidum to ofloxacin].

Treponema pallidum has not been yet cultivated. Hence any in vitro investigation is excluded, and it is owing to the experimental animal model, the rabbit, that we have studied the susceptibility of that germ to ofloxacin. This quinolone, owing to its pharmacokinetic and therapeutic properties, can specially be indicated in the treatment of Sexually Transmitted Diseases. Thus, its appeared to be of the utmost importance to know if the suggested schedule of treatment for STD, might not be susceptible to modify the course of a co-existing incubating syphilis by either delaying or inhibiting the apparition of the clinical features of primary syphilis. This study was undertaken at the incubation period, in syphilitic rabbits, using kinetic data obtained in man, after a given dosage of ofloxacin. Results were appraised upon converging data: lesions, bacteriology, and serology of the tested lot compared with two control batches of infected rabbits, the first one being untreated, the other having received the reference antibiotic treatment. From the data obtained and in the experimental settled conditions where this study was done, it results that ofloxacin has no effect on the course of the experimental syphilitic infection.

Adolescent↗
PubMed1982

Expression of Treponema pallidum antigens in Escherichia coli.

Treponema pallidum DNA was cloned in a bacteriophage. Clones were screened for expression of Treponema pallidum antigens by an in situ radioimmunoassay on nitrocellulose, with the use of subsequent reactions with syphilitic serum and radioiodinated Staphylococcus aureus protein A. One clone, which gave a strong signal, codes for at least seven antigens that react specifically with human antibodies to Treponema pallidum.

Antigens, Surface↗
PubMed1986

The antigenic interrelationship between the endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum Nichols strain. I. Treponemicidal activity of cross-reactive endoflagellar antibodies against T. pallidum.

Treponemicidal activity against Treponema pallidum, Nichols strain, by anti-endoflagellar antibodies and the presence of antigenic interrelationships between the endoflagella of Treponema phagedenis biotype Reiter (TPR) and T. pallidum have been demonstrated. SDS-PAGE profiles of purified endoflagella from both organisms were similar, identifying five polypeptide bands for TPR (37,000, 33,000 doublet, 30,000, and 27,000 daltons) and five polypeptide bands for T. pallidum (35,000, 33,000 doublet, 30,000, and 27,000 daltons). Antiserum against TPR endoflagella identified identical bands on Western blots of TPR, T. pallidum, and the respective endoflagellar preparations. Western blots confirmed the presence of antibodies in normal human serum (NHS) against the 33,000 dalton treponemal endoflagellar proteins. The complement-dependent treponemicidal activity of NHS against T. pallidum was completely removed by absorption with purified TPR endoflagella. Furthermore, rabbit antisera against TPR endoflagella were reactive in the Treponema pallidum immobilization (TPI) test. These findings demonstrate that anti-endoflagellar antibodies are treponemicidal against T. pallidum. A possible mechanism for this activity is discussed in relation to the subsurface location of endoflagella.

Animals↗
PubMed2006

[Expression and purification of membrane immunogens of Treponema pallidum in Pichia pastoris].

Genes of Treponema pallidum subsp. pallidum three membrane antigens (47kDa, 17kDa and 15kDa) were amplified by PCR with the template of the genomic DNA of Nichols strain and cloned into plasmid pPICZ B, the recombinant plasmids of pTP47, pTP17 and pTP15 were transformed into Pichia pastoris GS115. Recombinant antigens were expressed by the methanol induction and confirmed by the Western blot assay. Fusion antigens with 6 x His tag were purified using Ni-NTA agarose, and purified fusion protein yields were 4.8mg/L, 6.6mg/L and 25mg/L of cell culture for His-TP15, His-TP17 and His-TP47, respectively. Purity of all three antigens were more than 96% by SDS-PAGE assay. Particularly, His-TP17 was more about 6kDa molecular weight than that of expected probably because of glycosylation in Pichia pastoris. At last, these recombinant antigens were evaluated by ELISA assay with serum from syphilis patient and healthy blood donors, all three antigens showed strong sensitivity and specificity. So three membrane antigens of Treponema pallidum were expressed and purified fused with 6 x His tag in Pichia pastoris for the first time. The immunoreactivity results showed that all of which can be applied to diagnosis of Treponema pallidum, especially to diagnosis method based on combined two or three antigens.

Antigens, Bacterial↗
PubMed1981

Sequential changes in susceptibility to Treponema pallidum of rabbits previously infected with Treponema paraluis-cuniculi.

Rabbits immunised with virulent Treponema paraluis-cuniculi were challenged intradermally with graded doses of Treponema pallidum at three, five, seven, 12, and 30 months to ascertain the level of protection to T pallidum at various intervals after immunisation.Rabbits challenged at three months after immunisation showed no protection against T pallidum and developed syphilitic lesions significantly faster than the control rabbits, which suggests that the former rabbits were immunosuppressed. Some protection was evident at five and seven months after immunisation, as fewer inoculation sites developed syphilitic lesions with challenges of 10(3), 10(2), and 10 T pallidum and lesions developed significantly slower with 10(6) challenge. Two rabbits showed significant protection at 12 months after immunisation but a third, presumably still immunosuppressed, developed lesions significantly faster than the control rabbits after challenge. At 30 months after immunisation one rabbit was completely protected and developed no lesions after challenge; the other rabbit showed only partial protection against challenge with 10(4), 10(3), and 10(2) but complete protection against challenge with 10 T pallidum.T paraluis-cuniculi appeared to induce a state of immunosuppression by three months after infection; in one rabbit this may have been 12 months. In most immunised rabbits, however, limited cross-protection to low challenge doses of T pallidum developed by five months and was also detectable at seven and 12 months. Only one rabbit was completely resistant to challenge with 10(4)T pallidum after 30 months and another was only partly immune. Thus, T paraluis-cuniculi infection does not produce a rapid pronounced cross-immunity to T pallidum in rabbits, which may thus limit its usefulness as a vaccine against syphilis.

Animals↗
PubMed1982

Morphological destruction of cultured cells by the attachment of Treponema pallidum.

The incubation of Treponema pallidum with rabbit testicular cells, HEP-2 cells, human foreskin cells, rat cardiac cells, and rat skeletal muscle cells caused morphological disruption of these cultured cells. Control preparations of heat-inactivated treponemes, a high-speed supernatant in which treponemes had been pelleted, and culture medium failed to damage the tissue cells, as did viable treponemes when the cells were incubated in inverted Sykes-Moore chambers. Thus, cellular disruption is not associated with soluble treponemal, soluble inflammatory, or soluble testicular constituents but is mediated by the specific attachment of T pallidum. This organism apparently elaborates some type of toxic activity that lyses membranes: this may explain some of the histopathology of syphilitic disease.

Animals↗
PubMed2005

A monoclonal antibody that conveys in vitro killing and partial protection in experimental syphilis binds a phosphorylcholine surface epitope of Treponema pallidum.

Immunization with purified Treponema pallidum outer membrane vesicles (OMV) has previously resulted in high-titer complement-dependent serum bactericidal activity. In this study, OMV immunization resulted in the isolation of a monoclonal antibody, M131, with complement-dependent killing activity. Passive immunization of rabbits with M131 administered intravenously conferred significant immunity demonstrated by the failure of syphilitic lesions to appear at 29% of intradermal challenge sites (7/24) and a mean delay of approximately 8 days to lesion appearance at the remaining sites (17/24). M131 not only bound to OMV and to the surfaces of intact motile T. pallidum cells but also bound to organisms whose outer membranes were removed, indicating both surface and subsurface locations for the killing target. This target was determined to be a T. pallidum lipid. Lipid extracted from T. pallidum and made into liposomes bound M131. Reverse-phase high-pressure liquid chromatography separation and fraction collection mass spectrometry (LC-MS+) of T. pallidum lipid showed that the target of M131 was phosphorylcholine. M131 binding required both liposome formation and a critical concentration of phospholipid containing phosphorylcholine, suggesting that the epitope has both a conformational and a compositional requirement. M131 did not react with red blood cells, which have phosphorylcholine-containing lipids in their exterior membrane leaflets, or with Venereal Disease Research Laboratory antigen that also contains phosphorylcholine, further indicating the specificity of M131. This is the first physical demonstration of an antigen on the T. pallidum surface and indication that such a surface antigen can be a target of immunity.

Animals↗
PubMed1987

The hyaluronidase associated with Treponema pallidum facilitates treponemal dissemination.

Treponema pallidum contains hyaluronidase (Hase) associated with its surface. Experiments were performed to determine the functional role of this enzyme in syphilitic infection. The effects of incubating organisms with rabbit anti-bovine Hase or normal or immune sera were compared. Preincubation of treponemes with anti-Hase resulted in inhibition of treponemal degradation of hyaluronic acid, indicating that these antisera did in fact retard enzyme activity. Anti-Hase did not immobilize or neutralize T. pallidum. In addition, rabbits were immunized with bovine Hase and then challenged intradermally with organisms; subsequent lesion development was not affected. Anti-Hase did not block treponemal attachment to cultured testicular fibroblasts but did inhibit attachment to isolated capillaries. Rabbit amnions were used as an in vitro model for dissemination of T. pallidum. Anti-Hase retarded the penetration of organisms through the amnions. This inhibitory effect was dependent on the presence of amniotic hyaluronic acid. When this glycosaminoglycan was selectively removed, the anti-Hase lost its ability to inhibit treponemal penetration. When exogenous hyaluronic acid was added back to treated amnions, the inhibitory effect of anti-Hase was restored. Evans blue experiments were used to characterize treponeme-induced vascular leakage following intradermal inoculation of T. pallidum. Prior treatment of organisms with anti-Hase reduced dermal leakage of the dye, indicating the involvement of the treponemal Hase in causing vessel leakage. Finally, rabbit testicular infections were used as an in vivo model for dissemination; one testis was infected, and after 10 to 13 days, treponemes in the opposite testis were quantitated. The anti-Hase restricted dissemination of organisms. These findings point to the functional role of the treponemal Hase in facilitating disseminated syphilis.

Amnion↗
PubMed1982

Comparison of the morphology, antigenicity, and pathology of Nichols and Birmingham isolates of Treponema pallidum.

A strain of Treponema pallidum isolated from a homosexually acquired penile chancre in Birmingham in 1979 was serially passaged in rabbits and compared with the Nichols strain. The two strains appeared identical morphologically, and no gross differences were detected on antigenic analysis by two-dimensional immunoelectrophoresis. The Birmingham strain was, however, considerably less virulent for rabbits; the number of organisms required for equivalent time of development and severity of skin lesions was 10-fold to 100-fold greater for the Birmingham than for the Nichols strain. The pathology of testicular infection was broadly similar for both strains.

Animals↗