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.     

Dynamics of humoral immune response to Treponema pallidum proteins p17 and p41 at early stages of syphilis

In 60 blood sera from syphilis patients the titers of IgG to T. pallidum antigens p17 and p41 were detected with the use of the test system based on the recombinant analogues of T. pallidum proteins. The study revealed that primary syphilis was characterized by considerable prevalence of IgG to protein p41 with the total antibody level being low, while early latent syphilis was characterized mainly by considerable prevalence of IgG to protein p17 in the presence of high titers of antibodies. In secondary syphilis the sera contained a high total antibody level and a wide range of IgG ratios to individual antigens. On the basis of the data obtained the dynamics of immune response to antigens p17 and p41 at the early stages of the disease was hypothetically plotted. The curves of antibody levels had a wave-like character with the phase shifts of peaks for individual proteins and very low antibody titers (less than 1:100) in the negative peak areas. Conclusions were made that it was necessary to use the mixture of antigens in the production of the test systems and, when designing reference panels of sera, to include sera with extremely low titers of antibodies to individual proteins.     

免疫-PCR法检测梅毒螺旋体特异性抗体

以梅毒螺旋体重组蛋白为抗原,应用免疫-PCR方法检测梅毒螺旋体抗体,并同常规ELISA法进行比较,探讨免疫-PCR方法检测梅毒螺旋体特异性抗体的可行性。结果免疫-PCR法敏感性是常规ELISA法的104倍,阳性检出率高于ELISA法;对照血清标本梅毒螺旋体抗体检测为阴性。表明免疫-PCR方法具有较高敏感性和特异性,有一定的临床推广价值,对梅毒患者的早期诊断及时治疗等具有重要意义。     

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lacto-ferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.     

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.     

Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri

Miller, James N. (University of California School of Medicine, Los Angeles), J. H. De Bruijn, and J. H. Bekker. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamma-irradiated Treponema pallidum and Treponema reiteri. J. Bacteriol. 91:583-587. 1966.-Ultrasonic lysate preparations extracted from virulent Treponema pallidum, Nichols strain, suspensions exposed to 652,800 R of gamma-irradiation exhibited a loss in the serological reactivity of their heat-labile antigens; the heat-stable components of both the lysate and residue antigens were unaffected. The activity of heat-stable, cardiolipin T. pallidum complement-fixing antigen obtained from similarly irradiated organisms was also unaltered. gamma-Irradiation of the cultivable Treponema reiteri with dosages as high as 6,500,000 R failed to alter serologically either the heat-labile or heat-stable component of its lipopolysaccharide-protein (Reiter protein) antigen. The reactivity of the lipopolysaccharide portion of the Reiter protein complex with an antiserum to T. pallidum Nichols indicates previously unsuspected antigenic differences between the rabbit-adapted Nichols strain of the organism and so-called "wild" human strains of T. pallidum in which this antigen is generally absent.     

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.     

A novel beta-lactamase activity from a penicillin-binding protein of Treponema pallidum and why syphilis is still treatable with penicillin

Treponema pallidum, the causative agent of syphilis, is sensitive to penicillins. Yet, an abundant membrane-bound protein of this organism, Tp47, turns over penicillins. It is shown herein that the turnover process is a hydrolytic reaction that results in the corresponding penicilloates, products that have their beta-lactam bonds hydrolyzed. This is the reaction of beta-lactamases, bona fide resistance enzymes to beta-lactam antibiotics. Remarkably, the x-ray structure of Tp47 bears no resemblance to any other beta-lactamases or the related penicillin-binding proteins. Furthermore, evidence is presented that the reaction of Tp47 takes place in the absence of the zinc ion and does not involve intermediary acyl enzyme species. Hence, the beta-lactamase activity of Tp47 is the fifth known mechanism for turnover of beta-lactam antibiotics. Tp47 also exhibits a penicillin binding reaction, in the process of which the enzyme is covalently modified in the active site. The two reactions take place in two different active sites, and the events of the beta-lactamase activity are over 2,000-fold more rapid than the penicillin binding reaction. The level of beta-lactamase activity is high and is held back only by a strong product-inhibition component to the catalytic process. If natural selection would result in a mutant variant of Tp47 that overcomes product inhibition for the beta-lactamase activity, a novel bona fide resistance to penicillins will emerge in Treponema, which will be a disconcerting clinical development. The physiological functions of Tp47 are not known, but it is likely that this is at least a bifunctional enzyme involved in the processing of the Treponema peptidoglycan as a substrate.     

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.     

Isolation of the outer membranes from Treponema pallidum and Treponema vincentii.

The outer membranes from Treponema pallidum subsp. pallidum and Treponema vincentii were isolated by a novel method. Purified outer membranes from T. pallidum and T. vincentii following sucrose gradient centrifugation banded at 7 and 31% (wt/wt) sucrose, respectively. Freeze fracture electron microscopy of purified membrane vesicles from T. pallidum and T. vincentii revealed an extremely low density of protein particles; the particle density of T. pallidum was approximately six times less than that of T. vincentii. The great majority of T. vincentii lipopolysaccharide was found in the outer membrane preparation. The T. vincentii outer membrane also contained proteins of 55 and 65 kDa. 125I-penicillin V labeling demonstrated that t. pallidum penicillin-binding proteins were found exclusively with the protoplasmic cylinders and were not detectable with purified outer membrane material, indicating the absence of inner membrane contamination. Isolated T. pallidum outer membrane was devoid of the 19-kDa 4D protein and the normally abundant 47-kDa lipoprotein known to be associated with the cytoplasmic membrane; only trace amounts of the periplasmic endoflagella were detected. Proteins associated with the T. pallidum outer membrane were identified by one- and two-dimensional electrophoretic analysis using gold staining and immunoblotting. Small amounts of strongly antigenic 17- and 45-kDa proteins were detected and shown to correspond to previously identified lipoproteins which are found principally with the cytoplasmic membrane. Less antigenic proteins of 65, 31 (acidic pI), 31 (basic pI), and 28 kDa were identified. Compared with whole-organism preparations, the 65- and the more basic 31-kDa proteins were found to be highly enriched in the outer membrane preparation, indicating that they may represent the T. pallidum rare outer membrane proteins. Reconstitution of solubilized T. pallidum outer membrane into lipid bilayer membranes revealed porin activity with two estimated channel diameters of 0.35 and 0.68 nm based on the measured single-channel conductances in 1 M KCl of 0.40 and 0.76 nS, respectively.     

Assessment of the immune responses to Treponema pallidum Gpd DNA vaccine adjuvanted with IL-2 and chitosan nanoparticles before and after Treponema pallidum challenge in rabbits

Syphilis is a multistage,sexually transmitted disease caused by the spirochete,Treponema pallidum(Tp).A significantly high incidence of syphilis has been reported in several countries,including China,and there is an urgent need for the development of efficacious vaccines against syphilis.DNA vaccines are a major breakthrough in the field of vaccination with several advantages over traditional vaccines.Animal model studies of Tp DNA vaccines have not been reported elsewhere but our previous reports describe the development of a single-gene Tp DNA vaccine and preclinical immunization study.In this study,chitosan(CS) nanoparticles were used as a vector and an interleukin-2 expression plasmid(pIL-2) as an adjuvant to enhance a TpGpd DNA vaccine candidate(pTpGpd) in a rabbit Tp skin challenge model.At week 8 after the first immunization,three rabbits from each group were used to determine cytokine measurements and spleen lymphocyte proliferation assay.pTpGpd in combination with pIL-2 wrapped with CS led to the greatest enhancement of anti-TpGpd antibodies and T-cell proliferation.During infection,levels of anti-TpGpd antibodies and T-cell proliferation were measured.Both the serum special IgG and IL-2,interferon-γ were significantly increased by the co-injection of the IL-2 plasmid compared with the injection of TpGpd DNA alone(P<0.05).Furthermore,IL-2 plasmid coinjection efficiently enhanced the antigen-specific lymphocyte proliferation response.Additionally,the ratios of positive skin lesions and ulcer lesions in groups immunized with pTpGpd were significantly lower than those of the pIL-2,CS or pIL-2 mixed with CS control groups(P<0.001).CS vectored and pIL-2 adjuvanted pTpGpd immunized animals exhibited the lowest rates of positive skin tests(8.33%) and ulcer lesions(4.17%) and the fastest recovery(42 d).These experiments indicate that co-injection of a pIL-2 plasmid with pTpGpd DNA vaccine wrapped with CS can significantly strengthen the long-term stability of immune response during infection,efficiently improve the protective effect against T.pallidum spirochetes infection and attenuate syphilitic lesion development.     

Electron microscope study of the plasmid DNA from various strains of Treponema pallidum

Electron microscopic investigation and electrophoretic analysis of DNA from two cultural strains of pale treponemes (Treponema pallidum VIII and Treponema pallidum Reiter) allowed us to identify the circular molecules of plasmid DNA their size evaluated as 7 to 8 MD. Side by side with the molecules of this size the electron microscopic pictures of the plasmid DNA of both strains of pale treponemes show small superspiralized molecules of plasmid DNA. According to electrophoretic data the size of this DNA is evaluated as two thousand base pairs.     

Comparison of a recombinant-antigen enzyme immunoassay with Treponema pallidum hemagglutination test for serological confirmation of syphilis

A recombinant-antigen enzyme immunoassay (EIA), BioSCREEN anti-Treponema pallidum, was compared favorably with the T. pallidum hemagglutination test, in the detection of specific antibodies in different groups of sera from patients with primary (n = 38), secondary (n = 10), early latent (n = 28) and congenital syphilis (n = 2), patients with leptospirosis ( n= 8), infectious mononucleosis (n = 7), hepatitis (n = 9), diabetes mellitus (n = 11), rheumatoid arthritis (n = 13), leprosy (n = 11), tuberculosis (n = 9), HIV/Aids ( n= 12), systemic lupus erythematosus (n = 4), rheumatic fever (n = 3), old-persons (n = 9), pregnant women (n = 29) and blood donors (n = 164). The coincidence between them was 95.1%. The sensitivity and specificity of the EIA were 93.3% and 95.5%, respectively. Fifteen serum specimens belonging to old-persons, pregnant women, blood donors, and patients with human leptospirosis, hepatitis, diabetes mellitus, tuberculosis and rheumatic fever gave false-positive results by Venereal Disease Research Laboratory and/or Rapid Plasma Reagin. The EIA can be used as alternative method for the serological confirmation of syphilis.     

Circulating immune complexes in experimental syphilis and their relation to immunological response against Treponema pallidum

It was found that circulating immune complexes (CIC) were formed in rabbits at different times after infection with Treponema pallidum. The CIC which appeared at the beginning of the disease were short-lived (2-6 weeks) but those appearing later than 20 weeks after infection remained for 10-25 weeks. CIC contained both IgM and IgG classes of immunoglobulin. The antibodies present in CIC were found to be specific and nonspecific for T. pallidum. The presence of CIC led to a marked decline of treponemal antibodies in rabbit sera. The cell-mediated immune response measured by the macrophage migration inhibition (MMI) test at the beginning of the disease (up to 12 weeks) was not decreased. However, when syphilis lasted for more than 14 weeks and when CIC were formed mainly from IgG, a distinct decrease in the ability of lymphocytes to cause MMI was observed. These findings strongly suggest that IgG-complexes suppress the immunological responsiveness of lymphocytes against T. pallidum which in turn facilitates the multiplication of treponemes in the host.     

巢式PCR法在早期梅毒诊断中的应用评价

目的：评价巢式PCR（nPCR）法在早期梅毒诊断中的临床应用价值,以提高早期梅毒诊断的灵敏度和特异性。方法：选择2010年10月至2011年11月来我院就诊,经临床综合分析为一期梅毒的患者195例和同期就诊的120例非梅毒患者为研究对象,采用nPCR法对棉拭子标本和血液标本中梅毒螺旋体特异性基因tpp47进行扩增检测,所有标本同时做暗视野镜检和Tp—ELISA血清学检测。结果：nPCR法共检测出阳性标本176例,其灵敏度和特异性分别为90．3％和100％,明显高于暗视野镜检和Tp-ELISA法,差异有统计学意义。结论：nPCR法在早期梅毒诊断中具有较高灵敏度和特异性,可以作为暗视野镜检和血清学检测的补充试验。     

Serologic diagnosis of syphilis; the use of Treponema pallidum immobilization and immune adherence tests

TPI tests were carried out on 4,060 sera. Among 3,934 patients with reactive STS and no history of syphilis, 2,148 or 54.6 per cent had negative reaction to TPI tests, 1,695 or 43.1 per cent had positive reaction and 91 or 2.3 per cent had doubtful reaction. Two hundred and ninety-two or 73.0 per cent of 400 pregnant women with reaction to STS in the absence of a history of syphilis showed negative results by TPI test, 103 or 25.8 per cent had positive results and five or 1.2 per cent had doubtful reaction.Ninety-five or 75.4 per cent of 126 patients with a history of treated syphilis had positive reaction to TPI tests, 20 or 20.2 per cent had negative reaction and nine or 9.1 per cent had doubtful reaction.TPI and TPIA tests were done on 143 sera carefully selected for the study. Among 102 sera subjected to the TPI test, 46 or 100 per cent of these positive were also positive by TPIA tests, while 52 or 94.5 per cent of 55 TPI-negative sera were also nonreactive by TPIA test. One serum gave doubtful TPI test reaction and positive TPIA test reaction.     

Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.