Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.     

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.     

Segregation of B and T cell epitopes of Treponema pallidum repeat protein K to variable and conserved regions during experimental syphilis infection

Robust immune responses clear millions of treponemes to resolve lesions of primary and secondary syphilis, but cannot clear the treponemes that lead to debilitating and sometimes fatal tertiary syphilis. It is also known that the rabbit model and humans can be reinfected with heterologous isolates. How some treponemes are able to escape the immune system is unknown. In our laboratories rabbits immunized with the Seattle Nichols strain Treponema pallidum repeat protein K (TprK) were previously shown to have attenuated lesion development following challenge. In other isolates, TprK was shown to have seven discrete variable regions, with sequence variation among and within isolates. Using overlapping synthetic 20-aa peptides, we demonstrate that during experimental infection with the Nichols strain, the T cell responses are directed to conserved regions, while the Ab responses are directed primarily to variable regions. Abs from rabbits immunized with recombinant TprK recognized conserved and variable regions, suggesting that the conserved regions are inherently as immunogenic as the variable regions. TprK variability may allow some treponemes to escape recognition from Abs. The variable region heterogeneity may help explain the lack of protection against heterologous isolates.     

Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide research include continued elucidation of their structural locations and functional activities, identification and characterization of the low-abundance outer membrane proteins, further study of the immunoprotective and immunodiagnostic potential of T. pallidum proteins, and clarification of the roles of treponemal proteins in pathogenesis.     

Comparative investigation of the genomic regions involved in antigenic variation of the TprK antigen among treponemal species, subspecies, and strains

Although the three Treponema pallidum subspecies (T. pallidum subsp. pallidum, T. pallidum subsp. pertenue, and T. pallidum subsp. endemicum), Treponema paraluiscuniculi, and the unclassified Fribourg-Blanc treponeme cause clinically distinct diseases, these pathogens are genetically and antigenically highly related and are able to cause persistent infection. Recent evidence suggests that the putative surface-exposed variable antigen TprK plays an important role in both treponemal immune evasion and persistence. tprK heterogeneity is generated by nonreciprocal gene conversion between the tprK expression site and donor sites. Although each of the above-mentioned species and subspecies has a functional tprK antigenic variation system, it is still unclear why the level of expression and the rate at which tprK diversifies during infection can differ significantly among isolates. To identify genomic differences that might affect the generation and expression of TprK variants among these pathogens, we performed comparative sequence analysis of the donor sites, as well as the tprK expression sites, among eight T. pallidum subsp. pallidum isolates (Nichols Gen, Nichols Sea, Chicago, Sea81-4, Dal-1, Street14, UW104, and UW126), three T. pallidum subsp. pertenue isolates (Gauthier, CDC2, and Samoa D), one T. pallidum subsp. endemicum isolate (Iraq B), the unclassified Fribourg-Blanc isolate, and the Cuniculi A strain of T. paraluiscuniculi. Synteny and sequence conservation, as well as deletions and insertions, were found in the regions harboring the donor sites. These data suggest that the tprK recombination system is harbored within dynamic genomic regions and that genomic differences might be an important key to explain discrepancies in generation and expression of tprK variants among these Treponema isolates.     

The endemic treponematoses

Treponemal diseases comprise venereal syphilis (Treponema pallidum subsp. pallidum) and the endemic (non-venereal) treponematoses, i.e. yaws (T. pallidum subsp. pertenue), endemic syphilis (T. pallidum subsp. endemicum) and pinta (T. carateum). Treponemal diseases are distinguished on the basis of epidemiological characteristics and clinical manifestations. They are at present indistinguishable by morphological, immunological or serological methods. Several minor genetic differences have been identified among the subspecies. The endemic treponematoses have not yet been eliminated and are currently thought to affect at least 2.5 million persons. Renewed action towards the elimination of these diseases should be undertaken.     

Gene conversion: a mechanism for generation of heterogeneity in the tprK gene of Treponema pallidum during infection

The tprK gene sequence of Treponema pallidum subspecies pallidum (T. pallidum) is heterogeneous within and among isolates. Heterogeneity in the tprK open reading frame is localized in seven discrete variable (V) regions, and variability results from apparent base changes, insertions or deletions. The TprK V regions are the focus of anti-TprK antibodies arising during infection. To test our hypothesis that V region sequences change during infection and passage, we developed a clonal isolate from the Chicago strain of T. pallidum and confirmed V region diversification during passage of this isolate. We describe the sequence anatomy of the seven V regions of tprK and the identification of putative donor sites for new V region sequences, and we propose a model for generation of new V regions by segmental gene conversion. These findings suggest that antigenic variation of TprK occurs in T. pallidum and may be important in immune evasion and persistence.     

梅毒螺旋体疫苗的研究进展

梅毒是由密螺旋体苍白亚种( Treponema pallidum subsp. pallidum , Tp)感染引起的慢性系统性性传播疾病,流行于中低等收入国家。越来越多的临床病例表明清除Tp感染需要加强公共卫生筛查和治疗,而接种疫苗是预防Tp感染极有价值和首选的方法。本文概述了研制Tp疫苗的必要性,总结疫苗研究过程中候选抗原的相关信息及递送系统,分析Tp疫苗发展策略。     

Laboratory methods of diagnosis of syphilis for the beginning of the third millennium

Despite that the whole genome of T. pallidum, the causative agent of syphilis, has been sequenced, syphilis is, and will remain for some time, diagnosed by direct clinical observation and by laboratory methods. This review presents comprehensively most of the practical techniques used for direct detection of T. pallidum and lists all practical methods for phospholipid and treponemal antibodies detection. It describes most novel tests for syphilis, discusses problems with sero-creossreactivity in Lyme disease, immune responses in HIV-syphilis coinfected patients, and reviews serologic responses to antibiotic treatment.     

Stress proteins and the immune response to mycobacteria — Antigens as virulence factors?

Summary The immune response to mycobacterial infection includes pathogenic as well as protective activities. It is possible that different types of immune responses are associated with recognition of different antigenic determinants. Amongst the antigens which are prominent in antibody and T cell recognition of mycobacteria, we have identified members of highly conserved stress protein families. Mapping of antigenic determinants on stress proteins shows that both species-specific and conserved regions of these proteins can take part in immune recognition. Induction of an immune response to conserved, self-like, determinants on stress proteins could play a role in the immunopathology associated with chronic mycobacterial infections.     

Antigenic relatedness and N-terminal sequence homology define two classes of periplasmic flagellar proteins of Treponema pallidum subsp. pallidum and Treponema phagedenis

The periplasmic flagella of many spirochetes contain multiple proteins. In this study, two-dimensional electrophoresis, Western blotting (immunoblotting), immunoperoxidase staining, and N-terminal amino acid sequence analysis were used to characterize the individual periplasmic flagellar proteins of Treponema pallidum subsp. pallidum (Nichols strain) and T. phagedenis Kazan 5. Purified T. pallidum periplasmic flagella contained six proteins (Mrs = 37,000, 34,500, 33,000, 30,000, 29,000, and 27,000), whereas T. phagedenis periplasmic flagella contained a major 39,000-Mr protein and a group of two major and two minor 33,000- to 34,000-Mr polypeptide species; 37,000- and 30,000-Mr proteins were also present in some T. phagedenis preparations. Immunoblotting with monospecific antisera and monoclonal antibodies and N-terminal sequence analysis indicated that the major periplasmic flagellar proteins were divided into two distinct classes, designated class A and class B. Class A proteins consisted of the 37-kilodalton (kDa) protein of T. pallidum and the 39-kDa polypeptide of T. phagedenis; class B included the T. pallidum 34.5-, 33-, and 30-kDa proteins and the four 33- and 34-kDa polypeptide species of T. phagedenis. The proteins within each class were immunologically cross-reactive and possessed similar N-terminal sequences (67 to 95% homology); no cross-reactivity or sequence homology was evident between the two classes. Anti-class A or anti-class B antibodies did not react with the 29- or 27-kDa polypeptides of T. pallidum or the 37- and 30-kDa T. phagedenis proteins, indicating that these proteins are antigenically unrelated to the class A and class B proteins. The lack of complete N-terminal sequence homology among the major periplasmic flagellar proteins of each organism indicates that they are most likely encoded by separate structural genes. Furthermore, the N-terminal sequences of T. phagedenis and T. pallidum periplasmic flagellar proteins are highly conserved, despite the genetic dissimilarity of these two species.     

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.     

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.     

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.     

Monoclonal antibodies directed against major histocompatibility complex antigens bind to the surface of Treponema pallidum isolated from infected rabbits or humans

Evidence is presented for the association of class I major histocompatibility complex (MHC) antigens with the surface of Treponema pallidum during infection. A monoclonal antibody (IgG2a) directed against a murine H-2Kb epitope of public specificity reacted with the cell surface of T. pallidum, as assayed by the binding of protein A-colloidal gold in immunoelectron microscopy. Monoclonal antibodies directed against class I rabbit MHC antigens also reacted in immunofluorescence assays with material on the surface of rabbit-cultivated T. pallidum. In addition, impression smears of human syphilitic genital ulcers that were darkfield-positive for the presence of spirochetes were tested in immunofluorescence assays with monoclonal antibodies directed against human MHC antigens; antibody directed against HLA-ABC (class I) was reactive whereas antibody directed against HLA-DR (class II) was nonreactive. Results of the study suggest that the association of host-derived class I MHC antigens or molecular mimicry may play a role in T. pallidum evasion of host immune defenses.     

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.