Antigenic Structure of Treponema pallidum, Nichols Strain II. Extraction of a Polysaccharide Antigen with “Strain-specific” Serological Activity

An ultracentrifugally homogeneous heat-stable polysaccharide preparation free from serologically reactive rabbit testicular tissue antigen, including cardiolipin, was extracted from the Nichols strain of Treponema pallidum, and found to react by complement-fixation with homologous rabbit sera but not with human syphilitic sera. In addition, the reactive "strain-specific" component was found to be distinct from a second reactive component within the preparation related to an antigen of T. reiteri.     

Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens.

Genetic relationships among two strains of Treponema pallidum (Nichols and KKJ) and a strain of T. pertenue were determined by measuring the degree of deoxyribonucleic acid sequence homology. The results in indicated that these three virulent, noncultivable treponemes were genetically indistinguishable. Like T. pallidum (Nichols), T. pertenue (Gauthier) had no detectable deoxyribonucleic acid sequence homology with T. phagedenis (biotype Reiter), T. refringens (biotype Noguchi), or with salmon sperm.     

Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes.

Three genetically distinct groups of treponemes have been identified by saturation reassociation assays using 125I-labeled treponemal DNAs. The three groups are (i) virulent Treponema pallidum (Nichols strain), (ii) T. phagedenis and its biotypes Reiter and Kazan 5, and (iii) T. refringens biotypes Nichols and Noguchi. There is no detectable DNA sequence homology (less than 5%) among the three groups. The groups have distinct guanine + cytosine contents: 52.4 to 53.7% for T. pallidum, 41.5% for T. refringens, and 38 to 39% for T. phagedenis.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

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.     

Autoantibodies to creatine kinase in rabbits infected with Treponema pallidum

Sera from rabbits infected intratesticularly with Treponema pallidum (Nichols) for 30 days were examined for autoantibody reactivity against muscle and testis extracts by Western immunoblotting. Syphilitic sera (30 day) reacted with an autoantigen of 43,000 daltons in muscle extracts. The antigen was shown to be creatine kinase (CK). Studies with the use of an anti-CK ELISA showed that the autoantibody to CK first appeared 3 wk after infection, declined by 7 wk infection, and was absent in rabbits "mock"-infected with heat-killed T. pallidum. CK activity was not detected in sonicated or intact, washed T. pallidum, suggesting that the antibody was not produced in response to treponemal CK.     

Study of the plasmid composition of various strains of Treponema pallidum

Plasmid DNA has been isolated by soft alkaline and hard alkaline lysis from a pathogenic strain (Nichols) and two cultural strains (Reiter and VIII) of Treponema pallidum. Plasmid DNA was identified in all three strains. The molecular mass of identified plasmid DNA is 7 x 10(6) daltons according to the data of electrophoretic analysis in the agarose gel.     

Characterization of monoclonal antibodies to Treponema pallidum

Thirteen hybrid cell lines which produce mouse monoclonal antibodies to Treponema pallidum, the causative agent of syphilis, have been established. All of the monoclonal antibodies react with T. pallidum, Nichols strain, in ELISA and in immunofluorescence assays, but do not react with normal rabbit testicular tissue in the ELISA. Two of these antibodies were demonstrated to react with the nonpathogenic treponemes T. phagedenis, biotype Reiter, T. refringens (Noguchi strain), T. vincentii, and T. denticola (strains 11 and W), as well as with Borrelia recurrentis, Leptospira interrogans, serogroup Canicola, and the swine pathogen T. hyodysenteriae. The remaining 11 antibodies react with four recently isolated strains of T. pallidum, but with none of the related nonpathogens nor with Borrelia or Leptospira. Thus, our results to date indicate that these monoclonal antibodies may identify antigenic determinants that are specific either for T. pallidum alone or for those treponemes which are pathogenic for humans. The molecular specificities of six of the 13 antibodies were determined by Western blotting. We anticipate potential usefulness of these antibodies in the investigation of the antigenic structure of T. pallidum, the taxonomic study of the pathogenic and nonpathogenic treponemes, and in the diagnosis of syphilis.     

Comparative antigenic analysis of Treponema pallidum laboratory and street strains

The polypeptide and antigenic profiles of Treponema pallidum Nichols strain and two other more recently isolated 'street' strains of T. pallidum have been compared. PAGE and immunoblotting identified a 34.5 kDa polypeptide present in the Nichols strain which was absent from one of the other street strains. This polypeptide was shown to be associated with the axial filament in T. pallidum. Three other axial-filament-associated polypeptides of 37, 33 and 30 kDa were present in all strains examined. Axial filaments of all three strains were morphologically identical and all three strains were equally motile.     

Demonstration of rare protein in the outer membrane of Treponema pallidum subsp. pallidum by freeze-fracture analysis.

The surface of Treponema pallidum subsp. pallidum (T. pallidum), the etiologic agent of syphilis, appears antigenically inert and lacks detectable protein, as judged by immunocytochemical and biochemical techniques commonly used to identify the outer membrane (OM) constituents of gram-negative bacteria. We examined T. pallidum by freeze-fracture electron microscopy to visualize the architecture of its OM. Treponema phagedenis biotype Reiter (T. phagedenis Reiter), a nonpathogenic host-associated treponeme, and Spirochaeta aurantia, a free-living spirochete, were studied similarly. Few intramembranous particles interrupted the smooth convex and concave fracture faces of the OM of T. pallidum, demonstrating that the OM of this organism is an unusual, nearly naked lipid bilayer. In contrast, the concave fracture face of the OM of S. aurantia was densely covered with particles, indicating the presence of abundant integral membrane proteins, a feature shared by typical gram-negative organisms. The concentration of particles in the OM concave fracture face of T. phagedenis Reiter was intermediate between those of T. pallidum and S. aurantia. Similar to typical gram-negative bacteria, the OM convex fracture faces of the three spirochetes contained relatively few particles. The unique molecular architecture of the OM of T. pallidum can explain the puzzling in vitro properties of the surface of the organism and may reflect a specific adaptation by which treponemes evade the host immune response.     

梅毒螺旋体膜抗原基因在毕赤酵母中的表达和蛋白纯化

以梅毒螺旋体(Treponema pallidumsubsp.pallidum)Nichols菌株基因组DNA为模板,通过PCR扩增梅毒螺旋体47kDa、17kDa和15kDa 3个膜抗原基因,克隆进毕赤酵母表达载体pPICZ B,构建重组表达载体pTP47、pTP17、pTP15,转化酵母菌株GS115,甲醇诱导表达。表达菌体裂解后通过镍离子亲和层析获得3个抗原与6xHis tag的融合蛋白,重组蛋白的获得量分别为His-TP15:4.8mg/L;His-TP 17:6.6mg/L;His-TP47:25mg/L,经SDS-PAGE鉴定纯度都在96%以上,ELISA鉴定均具有很好的抗原性。从而首次在毕赤酵母中表达出梅毒螺旋体膜抗原,为梅毒血清学检测方法开辟了新的抗原制备途径。     

Footprint of positive selection in Treponema pallidum subsp. pallidum genome sequences suggests adaptive microevolution of the syphilis pathogen

In the rabbit model of syphilis, infection phenotypes associated with the Nichols and Chicago strains of Treponema pallidum (T. pallidum), though similar, are not identical. Between these strains, significant differences are found in expression of, and antibody responses to some candidate virulence factors, suggesting the existence of functional genetic differences between isolates. The Chicago strain genome was therefore sequenced and compared to the Nichols genome, available since 1998. Initial comparative analysis suggested the presence of 44 single nucleotide polymorphisms (SNPs), 103 small (≤3 nucleotides) indels, and 1 large (1204 bp) insertion in the Chicago genome with respect to the Nichols genome. To confirm the above findings, Sanger sequencing was performed on most loci carrying differences using DNA from Chicago and the Nichols strain used in the original T. pallidum genome project. A majority of the previously identified differences were found to be due to errors in the published Nichols genome, while the accuracy of the Chicago genome was confirmed. However, 20 SNPs were confirmed between the two genomes, and 16 (80.0%) were found in coding regions, with all being of non-synonymous nature, strongly indicating action of positive selection. Sequencing of 16 genomic loci harboring SNPs in 12 additional T. pallidum strains, (SS14, Bal 3, Bal 7, Bal 9, Sea 81-3, Sea 81-8, Sea 86-1, Sea 87-1, Mexico A, UW231B, UW236B, and UW249C), was used to identify "Chicago-" or "Nichols -specific" differences. All but one of the 16 SNPs were "Nichols-specific", with Chicago having identical sequences at these positions to almost all of the additional strains examined. These mutations could reflect differential adaptation of the Nichols strain to the rabbit host or pathoadaptive mutations acquired during human infection. Our findings indicate that SNPs among T. pallidum strains emerge under positive selection and, therefore, are likely to be functional in nature.     

Two 16S-23S ribosomal DNA intergenic regions in different Treponema pallidum subspecies contain tRNA genes

Abstract The 16S-23S intergenic spacers of Treponema pallidum subspecies pallidum , Nichols strain, and Treponema pallidum subspecies pertenue , Gauthier strain, have been cloned, characterized and sequenced. Isoleucine and alanine tRNA genes have been identified within the 16S-23S intergenic regions on separate alleles of 293 and 303 bases, respectively. The two alleles are present in both T.p. pallidum and T.p. pertenue , and show no sequence differences between the bacterial subspecies. The ile-tRNA and ala-tRNA genes show 65% and 84% sequence identity, respectively, with the homologous genes of the related spirochete, Borrelia burgdorferi .     

Conditions That Affect the Colorimetric Analysis of Lipopolysaccharide from Escherichia coli and Treponema pallidum

Material extracted from the Nichols nonpathogenic strain of Treponema pallidum by phenol-water was analyzed by employing a recently reported colorimetric test for detection of lipopolysaccharide (LPS). The fraction isolated from T. pallidum, in combination with the reagent dye, absorbed maximally at a wavelength in the range reported to be positive for LPS. Comparison of this reaction to that of a commercial preparation of Escherichia coli LPS revealed that time and temperature of incubation of the LPS-dye complexes were important variables which had marked but different effects on the LPS of the two sources. However, with careful control of these parameters, concentration-dependent standard curves were established for LPS of both sources. Our results indicate the cell wall of T. pallidum is similar to that of gram-negative organisms.     

Adoptive transfer of anti-syphilis immunity with lymphocytes from Treponema pallidum-infected guinea pigs

Spleen and lymph node cells taken from strain 2 and strain 13 guinea pigs at the peak of their primary immune response to cutaneous syphilitic infection could transfer partial protection to symptomatic disease to normal syngeneic recipients challenged with the Nichols strain of Treponema pallidum. These recipients of immune cells had significantly fewer treponemes disseminating to the regional lymph nodes and developed fewer and less severe cutaneous lesions that resolved faster than those in guinea pigs that had been infused with normal lymphoid cells. Immune donor cells also had the capacity to transfer specific delayed-type hypersensitivity responses for T. pallidum antigens. Both T and B cells were effective in conferring anti-syphilis immunity which was associated with the almost immediate development and persistence of substantially elevated levels of circulating anti-treponemal antibody in the protected recipients. Our findings in this adoptive transfer system provide the first direct experimental evidence implicating both cellular and humoral components of the immune response as important effector mechanisms in host resistance to the pathogenic spirochete causing venereal syphilis.     

Identification of Treponema pallidum penicillin-binding proteins.

Penicillin-binding proteins of 180, 89, 80, 68, 61, 41, and 38 kilodaltons were identified in Treponema pallidum (Nichols) by their covalent binding of [35S]benzylpenicillin. Penicillin-binding proteins are localized in the plasma membranes of many bacterial species and may serve as useful markers for determining plasma membrane intactness in T. pallidum fractionation studies.     

Demonstration of the in vitro phagocytosis of Treponema pallidum by rabbit peritoneal macrophages.

Evidence has been provided for the in vitro phagocytosis of virulent Treponema pallidum by stimulant-induced peritoneal macrophages. After the 4-hr incubation of macrophages with T. pallidum, treponemal antigens associated with the macrophages are specifically stained using indirect immunofluorescent techniques. Phagocytized treponemes appear within the cytoplasm of macrophages as round, brightly fluorescent "bodies" observable in increasing numbers as the duration of the treponeme-phagocyte interaction increases. Their presence is significantly reduced in the cytoplasm of macrophages that have been treated with cytochalasin B, a known inhibitor of phagocytosis, and in nonphagocytic fibroblasts. Additionally, supportive evidence for T. pallidum phagocytosis in vitro has been provided by electron microscopic examination in which treponemes have been demonstrated within typical phagocytic vacuoles. This study also provides evidence that immune serum factor(s) significantly promote the phagocytosis of T. pallidum, although a contribution by heat-labile serum factors has not been demonstrated. The possible mechanisms of immune serum contribution and the implications of the demonstration of T. pallidum phagocytosis are discussed.     

Sequence diversity of Treponema pallidum subsp. pallidum tprK in human syphilis lesions and rabbit-propagated isolates

The tprK gene of Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, belongs to a 12-member gene family and encodes a protein with a predicted cleavable signal sequence and predicted transmembrane domains. Except for the Nichols type strain, all rabbit-propagated isolates of T. pallidum examined thus far are comprised of mixed populations of organisms with heterogeneous tprK sequences. We show that tprK sequences in treponemes obtained directly from syphilis patients are also heterogeneous. Clustering analysis demonstrates that primary chancre tprK sequences are more likely to cluster within a sample than among samples and that tighter clustering is seen within chancre samples than within rabbit-propagated isolates. Closer analysis of tprK sequences from a rabbit-propagated isolate reveals that individual variable regions have different levels of diversity, suggesting that variable regions may have different intrinsic rates of sequence change or may be under different levels of selection. Most variable regions show increased sequence diversity upon passage. We speculate that the diversification of tprK during infection allows organisms to evade the host immune response, contributing to reinfection and persistent infection.     

Antigenic Relationships of 14 Treponemes Demonstrated by Immunofluorescence

Immunofluorescence was used as an aid in the antigenic grouping of 14 cultivable treponemes. Antisera were prepared versus each treponemal strain, and the antiglobulins were conjugated with fluorescein isothiocyanate. A common antigen-antibody system, detected in the strains studied, was removed by absorption of each conjugate with Reiter or Borrelia vincentii treponemes. Thus, five categories based on shared group-specific antigens were revealed. Serogroup I: Reiter, English Reiter, Kazan, Kazan numbers 2, 4, 5, and 8. Serogroup II: Nichols and Noguchi. Serogroup III: three oral treponemes, MRB, FM, and N-39. Serogroup IV: B. vincentii. Serogroup V: Treponema zuelzerae. The five serogroups apparently are related by an immunofluorescent common antigen.