Pathogen-related oral spirochetes from dental plaque are invasive.

Spirochetes that share pathogen-restricted antigens with Treponema pallidum subsp. pallidum have been identified in dental plaque and diseased gingival tissues, but it is not known whether these spirochetes possess virulence characteristics. In this study, plaque spirochetes were able to transmigrate a tissue barrier in vitro and were identified on the other side by using monoclonal antibodies specific for pathogen-restricted determinants from T. pallidum subsp. pallidum. This invasive capability is shared with T. pallidum subsp. pallidum, but cultured oral and intestinal treponemes did not perforate the tissue barrier. Cocultures indicated that invasive treponemes do not create opportunities for cultivable oral treponemes to cross the barrier. These findings indicate that gingival tissues may be a port of entry for previously unrecognized invasive spirochetes in humans.     

Immunochemical analysis of serum-related proteins in the respiratory tract secretions of normal mice.

The interaction of Treponema pallidum (Nichols strain) with cultured cells was investigated under aerobic conditions. Cell monolayers derived from rabbit testicular tissue extended the survival of treponemes as indicated by active motility. Large numbers of organisms rapidly attached to cultured cells. Within 3 h, one to twelve actively motile treponemes were attached to 25 to 50% of the cells. In addition, T. pallidum attained intracellularity as early as 30 min after inoculation of the cell monolayers. In sharp contrast, T. phagedenis biotype Reiter and T. denticola did not attach and did not enter cultured cells. Most importantly, intracellular and/or attached T. pallidum retained virulence for at least 24 h. Similar observations of attachment and retention of virulence were detected with ME-180, a cell line derived from a human cervical carcinoma. Preliminary studies with superoxide dismutase indicated that this enzyme prolonged treponemal motility and retention of virulence in the presence of cultured cells. These data provide guidelines for further investigations of in vitro cultivation of T. pallidum.     

Conservation of the 15-kilodalton lipoprotein among Treponema pallidum subspecies and strains and other pathogenic treponemes: genetic and antigenic analyses.

The 15-kDa lipoprotein of Treponema pallidum is a major immunogen during natural syphilis infection in humans and experimental infection in other hosts. The humoral and cellular immune responses to this molecule appear late in infection as resistance to reinfection is developing. One therefore might hypothesize that this antigen is important for protective immunity. This possibility is explored by using both genetic and antigenic approaches. Limited or no cross-protection has been demonstrated between the T. pallidum subspecies and strains or between Treponema species. We therefore hypothesized that if the 15-kDa antigen was of major importance in protective immunity, it might be a likely site of antigenic diversity. To explore this possibility, the sequences of the open reading frames of the 15-kDa gene have been determined for Treponema pallidum subsp. pallidum (Nichols and Bal-3 strains), T. pallidum subsp. pertenue (Gauthier strain), T. pallidum subsp. endemicum (Bosnia strain), Treponema paraluiscuniculi (Cuniculi A, H, and K strains), and a little-characterized simian isolate of Treponema sp. (Fribourg-Blanc strain). No significant differences in DNA sequences of the genes for the coding region of the 15-kDa antigen were found among the different species and subspecies studied. In addition, all organisms showed expression of the 15-kDa antigen as determined by monoclonal antibody staining. The role of the 15-kDa antigen in protection against homologous infection with T. pallidum subsp. pallidum Nichols was examined in rabbits immunized with a purified recombinant 15-kDa fusion protein. No alteration in chancre development was observed in immunized, compared to unimmunized, rabbits, and the antisera induced by the immunization failed to enhance phagocytosis of T. pallidum subsp. pallidum by macrophages in vitro. These results do not support a major role for this antigen in protection against syphilis infection.     

Treponema pallidum infection in subcutaneous polyethylene chambers in rabbits.

Male New Zealand white rabbits with subcutaneous polyethylene chambers in place for at least 3 months were inoculated by one of the following three methods: (i) "intra-chamber" (IC) inoculation with "normal" chamber fluid: (ii) intratesticular inoculation with Treponema pallidum; or (iii) IC inoculation with T. pallidum. Rabbits given dexamethasone only, oxisuran only, both drugs, or no drug were observed serially after inoculation. T. pallidum survived and temporarily multiplied to significant numbers within subucutaneous chambers after IC inoculation in rabbits given dexamethasone. In rabbits not treated with dexamethasone, T. pallidum counts in chamber fluid decreased rapidly and remained at low levels for 30 days after IC inoculation. Oxisuran appeared to have little or no effect on T. pallidum multiplication. All rabbits studied had a nonreactive serum and chamber fluid serological test for syphilis before inoculation. All rabbits inoculated with T. pallidum eventually developed reactive serum and chamber fluid serological tests. The IC route of inoculation was associated with a delay in the development of serum serological reactivity and with earlier chamber fluid reactivity as compared with the intratesticular route of inoculation. An immediate but transent influx of polymorphonuclear leukocytes was associated with IC inoculation of T. pallidum. Chamber fluid total protein content declined very slightly in all groups of rabbits during the month after inoculations. Successful cultivation of T. pallidum in an in vivo setting suggests that this animal model may be useful in further studies of the biology of the organism of the pathogenesis, immunology, and treatment of syphilis.     

Specific immunofluorescence staining of Treponema pallidum in smears and tissues.

To date, tissue sections prepared from Formalin-fixed tissues have not been successfully stained with Treponema pallidum subspecies-specific antibody in a direct fluorescent-antibody assay. While current methods stain T. pallidum, they do not distinguish T. pallidum from other spirochetes such as Borrelia burgdorferi (E. F. Hunter, P. W. Greer, B. L. Swisher, A. R. Simons, C. E. Farshy, J. A. Crawford, and K. R. Sulzer, Arch. Pathol. Lab. Med. 108:878-880, 1984). Because trypsin pretreatment of tissue sections has enhanced other immunofluorescent-antibody (IFA) applications, we compared the use of the trypsin digestion method with the current 1% ammonium hydroxide (NH4OH) method as a means to obtain specific staining of T. pallidum in tissues by both direct and indirect IFA techniques. Pretreated T. pallidum-infected tissues sections from rabbits, hamsters, and humans were quantitatively examined with the direct fluorescent-antibody-T. pallidum test conjugate absorbed with Treponema phagedenis, the Reiter treponeme. For indirect staining, a serum specimen from a patients with syphilis absorbed by affinity chromatography with T. phagedenis was used as the primary reagent, and a fluorescein isothiocyanate-labeled rabbit anti-human globulin was used as the secondary reagent. Serum specificity was established first by examining antigen smears of T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, B. burgdorferi, T. phagedenis, and Treponema denticola MRB and then by examining tissues infected with these pathogens plus those infected with four Leptospira serovars. When we stained tissue using the direct IFA method that is currently a standard method for the examination of chancre smears, we found it to be unsuitable for use with tissue. Trypsin digestion did not offer an improvement over the NH4OH pretreatment method in the specific identification of T. pallidum by direct IFA. However, specific identification of T. pallidum in tissue sections was obtained by the indirect IFA technique after either trypsin or NH4OH pretreatment.     

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.     

Relationship of Treponema pallidum to Acidic Mucopolysaccharides

Attempts were made to relate Treponema pallidum to the acidic mucopolysaccharides that occur in vivo within host ground substance and in vitro on the surface of cultured testicular cells. Infected testicular tissue was fixed and processed for transmission electron microscopy in the presence of ruthenium red. The use of this inorganic dye demonstrated the large quantity of mucopolysaccharide within testicular tissue and the intimate association of treponemes with this material. Wheat germ agglutinin and soybean agglutinin agglutinated freshly harvested trypsinized testicular cells and trypsinized cultured cells derived from normal rabbit testes (NRT). When stained with toluidine blue, both cell preparations were metachromatic. Prior treatment of cultured NRT cells with hyaluronidase slightly decreased their sensitivity to agglutination by wheat germ agglutinin and soybean agglutinin. Lectin agglutination, metachromasia, and hyaluronidase susceptibility indicated that freshly harvested testicular cells and NRT cells have surface-associated acidic mucopolysaccharides that are probably hyaluronic acid and chondroitin sulfate. A rabbit erythrocyte "sandwich" technique was devised to show that hyaluronidase removed wheat germ agglutinin receptors from the cultured NRT cells. Prior incubation of NRT cells with hyaluronidase, followed by the addition of T. pallidum, resulted in a reduction in numbers of treponemes attached to the NRT cells. The attachment of T. pallidum appears to be mediated through the acidic mucopolysaccharides on the surface of NRT cells. The findings are discussed in terms of the importance of host ground substance mucopolysaccharide to the syphilitic infective process.     

A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.     

Sensitive detection of Treponema pallidum by using the polymerase chain reaction.

We have developed a sensitive assay for Treponema pallidum subsp. pallidum (T. pallidum), the agent of veneral syphilis, based upon the polymerase chain reaction (PCR). A 658-bp portion of the gene encoding the 47-kDa membrane immunogen was amplified, and the PCR products were probed by DNA-DNA hybridization with a 496-bp fragment internal to the amplitifed DNA. The assay detected approximately 0.01 pg of purified T. pallidum DNA, and positive results were obtained routinely from suspensions of treponemes calculated to contain 10 or more organism and from some suspensions calculated to contain a single organism. Specific PCR products were obtained for the closely related agent of yaws, Treponema pallidum subsp. pertenue, but not with human DNA or DNAs from other spirochetes (including Borrelia burgdoferi), skin microorganisms, sexually transmitted disease pathogens, and central nervous system pathogens. T. pallidum DNA was detected in serum, cerebrospinal fluids, and amniotic fluids from syphilis patients but not in in nonsyphilitic controls. T. pallidum DNA was also amplified from paraffin-embedded tissue. The diagnosis of syphillis by using PCR may become a significant addition to the diagnostic armamentarium and a valuable technique for the investigation of syphilis pathogenesis.     

Immune response to Treponema pertenue and Treponema pallidum Nichols in patients with yaws.

Human sera from African patients with acute yaws were analysed by Western blot (WB) against antigens of Treponema pallidum Nichols and two Treponema pertenue isolates. The Western blot patterns were remarkably similar from one patient to another, and strains of both subspecies exhibited exactly the same banding pattern. Sera from yaws patients failed to detect at least one antigen in T. pertenue which was absent from T. pallidum.     

Antigenic cross-reactivity between Treponema pallidum and other pathogenic members of the family Spirochaetaceae.

The antigenic cross-reactivity between Treponema pallidum and several pathogenic members of the family Spirochaetaceae was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques. Blots of T. pallidum antigens were incubated with antiserum from rabbits infected or immunized with T. pallidum, Treponema paraluiscuniculi, Treponema hyodysenteriae (strains B204 and T22), Borrelia hermsii serotype 7, or Leptopsira interrogans serogroup Canicola. T. pallidum contained 22 antigenic molecules ranging from 85,000 to 12,000 daltons which were recognized by serum from rabbits infected with T. pallidum. Serum from rabbits infected with T. paraluiscuniculi cross-reacted with 21 of these molecules and faintly reacted with a band at 15,000 daltons which was not recognized by anti-T. pallidum serum. Antisera directed against strains B204 and T22 of T. hyodysenteriae cross-reacted with 11 and 10 antigens of T. pallidum, respectively. B. hermsii and L. interrogans serogroup Canicola antisera detected 11 and 10 treponemal antigens, respectively. Many of the T. pallidum antigens detected by antisera against T. hyodysenteriae, B. hermsii, or L. interrogans serogroup Canicola have been previously identified as containing moieties also found on the nonpathogenic Treponema phagedenis, biotype Reiter, and may therefore represent group antigens common to members of the family Spirochaetaceae.     

Host response to Treponema pallidum infection. III. Demonstration of autoantibodies to heart in sera from infected rabbits.

Sera from rabbits infected intratesticularly with Treponema pallidum but not from animals injected intratesticularly with other bacteria or with extract of normal rabbit testes demonstrated autoantibody to heart tissue. The antibody was organ-specific with cross-reactivity to skeletal muscle but was not species-specific. It could not be absorbed by T. pallidum, T. reiteri, Veneral Disease Research Laboratory reagent or rabbit mitochondrial preparation. The antibody had a transitional pattern of appearance; it could be demonstrated between 30 and 60 days after infection but it decreased or disappeared thereafter. In many instances, it could be shown between 2 and 3 years after infection. The finding of the heart-reacting antibody strongly suggests an autoimmune phenomenon associated with T. pallidum infection.     

Antigenic interrelationship between endoflagella of Treponema phagedenis biotype Reiter and Treponema pallidum (Nichols): molecular characterization of endoflagellar proteins.

Purified endoflagella from nonpathogenic Treponema phagedenis biotype Reiter were characterized biochemically and compared antigenically with the endoflagellar proteins of Treponema pallidum. T. phagedenis biotype Reiter endoflagella were dissociated into constituent polypeptides by incubation under conditions which disrupt noncovalent bonds. Chymotrypsin peptide maps of T. phagedenis biotype Reiter endoflagellar proteins revealed that the 37- and 33-kilodalton (kDa) major components shared significant homology with the 27- and 30-kDa minor components, respectively. The peptide maps also suggested that the major components shared a lesser degree of structural similarity with each other. These relationships were confirmed by Western blots of T. phagedenis biotype Reiter endoflagellar proteins employing antibodies that were purified against the individual endoflagellar polypeptides. Western blots of T. pallidum with the purified antibodies also demonstrated strong cross-reactivity between the T. phagedenis biotype Reiter endoflagellar proteins and T. pallidum proteins of identical or similar molecular weights. A unique Western blotting technique that we called epitope bridging was used to determine that the 37-kDa subunit contains most of the external epitopes on T. phagedenis biotype Reiter endoflagella. Immunoelectron microscopy with human syphilitic serum and rabbit T. phagedenis biotype Reiter endoflagellar antiserum confirmed the presence of cross-reactive epitopes on the surface of intact T. phagedenis and T. pallidum endoflagella.     

Immunofluorescent staining of Treponema in tissues fixed with formalin

Immunofluorescent examination of formalin-fixed tissue for Treponema pallidum has generally been unsatisfactory because of nonspecific background fluorescence and poor contrast. We examined the process of treating deparaffinized formalin-fixed tissue sections with 1% ammonium hydroxide (NH4OH) to improve fluorescent staining. Treponema pallidum- and Treponema pertenue-infected rabbit testes or human tissue biopsy specimens fixed in 10% buffered formalin and embedded in paraffin were examined. Sections were cut one week to five years after embedment. Tissues were then stained with fluorescein- or rhodamine-labeled human anti- T pallidum globulin for 30 minutes at 37 degrees C. Treponemes were consistently stained and background staining was generally reduced after NH4OH treatment in both fresh and stored tissue. Cutting sections at a thickness of approximately 2 micron was critical to achieve optimal fluorescence.     

In vitro cultivation of Treponema pallidum: independent confirmation.

In vitro cultivation of the virulent Nichols strain of Treponema pallidum was achieved in a tissue culture system as described by A. H. Fieldsteel, D. L. Cox, and R. A. Moeckli (Infect. Immun. 32:908-915, 1981). In 7 of 8 experiments, 8.9- to 26.2-fold increases in the number of T. pallidum were observed over a 12- to 12-day period of incubation.     

Intracellular Location of Treponema pallidum (Nichols Strain) in the Rabbit Testis

During investigations designed to obtain purified suspensions of virulent Treponema pallidum (Nichols strain), infected rabbit testicular tissue was routinely examined in the electron microscope. Morphologically typical T. pallidum were found intracellularly within the cytoplasmic substance of fibroblasts, interstitial and Leydig cells, and of spermatocytes. The importance of these observations to latency and treatment is discussed.     

Assessment of the kinetics of Treponema pallidum dissemination into blood and tissues in experimental syphilis by real-time quantitative PCR

Little is known about the size and kinetics of treponemal burdens in blood and tissues during acquired or experimental syphilitic infection. We used real-time quantitative PCR to measure Treponema pallidum DNA levels in rabbits infected intratesticularly with the prototype Nichols strain. At the outset, we performed a series of in vitro blood spiking experiments to determine the effect of blood processing procedures on the distribution of treponemes in various blood components. T. pallidum DNA levels in plasma and whole blood were approximately 10-fold higher than those in serum and more than 200-fold greater than those in peripheral blood mononuclear cells (PBMCs). Ten rabbits were inoculated intratesticularly with doses of treponemes ranging from 4 x 10(7) to 2 x 10(8) organisms. In five rabbits, T. pallidum DNA levels were measured sequentially in serum, plasma, whole blood, and PBMCs until sacrifice at peak orchitis, at which time brain, kidney, liver, spleen, and testicles were harvested; blood and organs were also harvested at orchitis from the other five rabbits. T. pallidum DNA was detected in plasma within 24 h postinfection. Treponeme levels in whole blood and blood components increased significantly with the development of peak orchitis. Overall, levels in serum and PBMCs were lower than those in plasma and whole blood; this disparity was particularly marked at early time points. Significantly greater numbers of spirochetes were found in the spleen than in liver, kidney, or brain tissue at the time of sacrifice. Our findings highlight the remarkable capacity of T. pallidum to disseminate from the site of infection to blood and tissues, and they identify the spleen as a prime target for treponemal invasion.     

BAC library of T. pallidum DNA in E. coli

Treponema pallidum subspecies pallidum (Nichols) chromosomal DNA was used to construct a large insert bacterial artificial chromosome (BAC) library in Escherichia coli DH10B using the pBeloBAC11 cloning vector; 678 individual insert termini of 339 BAC clones (13.9 x coverage) were sequenced and the cloned chromosomal region in each clone was determined by comparison to the genomic sequence. A single 15.6-kb region of the T. pallidum chromosome was missing in the BAC library, between bp 248727 and 264323. In addition to the 12 open reading frames (ORFs) coded by this region, one additional ORF (TP0596) was not cloned as an intact gene. Altogether, 13 predicted T. pallidum ORFs (1.25% of the total) were incomplete or missing in the library. Three of 338 clones mapped by restriction enzyme digestion had detectable deletions and one clone had a detectable insertion within the insert. Of mapped clones, 19 were selected to represent the minimal set of E. coli BAC clones covering 1026 of the total 1040 (98.7%) predicted T. pallidum ORFs. Using this minimal set of clones, at least 12 T. pallidum proteins were shown to react with pooled sera from rabbits immunized with T. pallidum, indicating that at least some T. pallidum genes are transcribed and expressed in E. coli.     

Monoclonal antibody analysis of specific antigenic similarities among pathogenic Treponema pallidum subspecies

Murine monoclonal antibodies directed against a 47,000-dalton immunodominant surface-exposed antigen of Treponema pallidum subsp. pallidum (Nichols) were isolated. These monoclonal antibodies cross-reacted with analogous 47,000-dalton antigens of two other virulent treponemes, T. pallidum subsp. pertenue and T. pallidum subsp. endemicum (Bosnia A), as determined by radioimmunoassay and immunoblot analyses. Immunoelectron microscopy confirmed that the 47,000-dalton antigen of T. pallidum subsp. pallidum was a surface-associated cellular component. Surface binding assays and immunoelectron microscopic studies also suggested that the analogous 47,000-dalton antigenic component of T. pallidum subsp. pertenue may not have been oriented toward the bacterial surface in the same way as the T. pallidum subsp. pallidum antigen or that the relevant antigenic determinant(s) may not have been exposed to the outer surface in the same way. The significance of this antigen relative to its apparent conservation among pathogenic treponemes and its possible diagnostic and vaccinogenic potentials are discussed.     

Treponema phagedenis encodes and expresses homologs of the Treponema pallidum TmpA and TmpB proteins.

We cloned and sequenced the genes from Treponema phagedenis Kazan 5 encoding proteins homologous to the TmpA and TmpB proteins of Treponema pallidum subsp. pallidum Nichols (hereafter referred to as T. pallidum). Although previous reports suggested that the TmpA and TmpB proteins were specific for T. pallidum, we found that homologs for both were expressed in T. phagedenis Kazan 5 and Reiter. The TmpA protein from T. phagedenis contained the consensus sequence that bacterial lipoproteins require for posttranslational modification and subsequent proteolytic cleavage by signal peptidase II and showed 42% amino acid sequence identity with the TmpA protein from T. pallidum. The TmpB proteins of T. phagedenis and T. pallidum had similar amino acid sequences at their amino- and carboxy-terminal ends. The central portions of both of these proteins contained four repeats of the amino acid sequence EAARKAAE. The TmpB protein from T. phagedenis had an additional amino acid sequence repeat (consensus sequence KAAKE/D) that was not found in the TmpB protein from T. pallidum; this repeat was most remarkable, as it occurred 17 times in succession. These repeated amino acid sequences probably created an extensive alpha-helix region within the TmpB proteins. As with T. pallidum, the stop codon of the T. phagedenis tmpA gene overlapped the start codon of its tmpB gene. Northern blot analysis showed that the T. phagedenis tmpA and tmpB genes were probably transcribed into a single 2.5-kb mRNA molecule. Western blot (immunoblot) analysis demonstrated that both proteins were expressed by T. phagedenis. The high degree of amino acid sequence conservation seen with the TmpA and TmpB proteins from two different Treponema species suggests that they may play crucial roles in the biology of these organisms.