Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp. pallidum.

Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp. pallidum (T. pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M. V. Norgard, N. R. Chamberlain, M. A. Swancutt, and M. S. Goldberg, Infect. Immun. 54:500-506, 1986). To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties. Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T. pallidum DNA fragment. A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system. Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular masses were observed that were identical to those previously detected on Western blots of native T. pallidum antigens with monoclonal antibodies. Sarkosyl extraction of E. coli recombinant cell envelopes localized the 47-kDa protein to both the inner and outer membranes of E. coli. The absolute requirement of detergents (N-lauroylsarcosine, 3-[(3-chloramidopropyl)dimethylammonio]-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40) for solubilization of the antigen from E. coli cell envelopes and the observation that the recombinant protein partitioned into the detergent phase on Triton X-114 solubilization were consistent with the fact that it is a hydrophobic, integral membrane protein. Western blots of the 47-kDa antigen purified by immunoaffinity chromatography supported results of previous reports that the 47-kDa protein is specific to pathogenic treponemes.     

Molecular cloning and characterization of the 15-kilodalton major immunogen of Treponema pallidum.

Pathogen-specific membrane immunogens of Treponema pallidum subsp. pallidum (T. pallidum) have been identified previously by phase partitioning with the nonionic detergent Triton X-114. One of these antigens, a 15-kilodalton (kDa) polypeptide, is expressed in relatively small quantities in T. pallidum but is highly immunogenic in both human and experimental syphilis. The native T. pallidum antigen was purified to homogeneity from the mixture of Triton X-114 detergent-phase proteins by chromatofocusing. Recombinant Escherichia coli clones were selected from a T. pallidum genomic DNA library by using monoclonal antibodies specific to the 15-kDa antigen; immunoblotting and minicell analyses confirmed expression of the 15-kDa protein in the transformants. Southern hybridization with a 1.1-kilobase fragment of DNA encoding the 15-kDa-antigen gene indicated that the gene is probably present in a single copy within the genomes of both T. pallidum and T. pallidum subsp. pertenue (the agent of yaws), while it is absent from the genome of the nonpathogenic Treponema phagedenis biotype Reiter. Cell fractionation studies with Triton X-114 demonstrated that the recombinant polypeptide possesses hydrophobic properties similar to those of the native antigen and localized the cloned 15-kDa antigen to the inner membrane of E. coli. Protein processing experiments in minicells revealed that a precursor appears to be processed to the mature 15-kDa polypeptide.     

Molecular cloning and expression of Treponema pallidum DNA in Escherichia coli K-12.

A gene bank of Treponema pallidum DNA in Escherichia coli K-12 was constructed by cloning SauI-cleaved T. pallidum DNA into the cosmid pHC79. Sixteen of 800 clones investigated produced one or more antigens that reacted with antibodies from syphilitic patients. According to the separation pattern of the antigens produced on sodium dodecyl sulfate-polyacrylamide gels, six different phenotypes were distinguished among these 16 clones. These antigens reacted also with anti-T. pallidum rabbit serum. No antibodies against the cloned antigens were found in normal rabbit serum and in nonsyphilitic human serum. The antigens produced by the E. coli K-12 recombinant DNA clones comigrated in sodium dodecyl sulfate-polyacrylamide gels with antigens extracted from T. pallidum bacteria, suggesting that the treponemal DNA is well expressed in E. coli K-12. Several of the cosmid recombinant plasmids have been subcloned, resulting in smaller T. pallidum recombinant plasmids which are more stably maintained in the cell and produce more treponemal antigen. Monoclonal antibodies were raised against T. pallidum, and one hybridoma produced antibodies that reacted not only with an antigen from T. pallidum but also with the antigen produced by one of the E. coli clones.     

Identification and characterization of the Treponema pallidum tpn50 gene, an ompA homolog.

Treponema pallidum is a pathogenic spirochete that has no known genetic exchange mechanisms. In order to identify treponemal genes encoding surface and secreted proteins, we carried out TnphoA mutagenesis of a T. pallidum genomic DNA library in Escherichia coli. Several of the resulting clones expressed enzymatically active T. pallidum-alkaline phosphatase fusion proteins. The DNA sequence of the 5' portion of a number of the treponemal genes was obtained and analyzed. A recombinant clone harboring plasmid p4A2 that encoded a treponemal protein with an approximate molecular mass of 50,000 Da was identified. Plasmid p4A2 contained an open reading frame of 1,251 nucleotides that resulted in a predicted protein of 417 amino acids with a calculated molecular mass of 47,582 Da. We have named this gene tpn50 in accordance with the current nomenclature for T. pallidum genes. A 1.9-kb HincII-ClaI fragment from p4A2 that contained the tpn50 gene was subcloned to produce p4A2HC2. Comparison of the predicted amino acid sequence of TpN50 with protein sequences in the National Center for Biotechnology Information data base indicated statistically significant homology to the Pseudomonas sp. OprF, E. coli OmpA, Bordetella avium OmpA, Neisseria meningitidis RmpM, Neisseria gonorrhoeae PIII, Haemophilus influenzae P6, E. coli PAL, and Legionella pneumophila PAL proteins. These proteins are all members of a family of outer membrane proteins that are present in gram-negative bacteria. The tpn50 gene complemented E. coli ompA mutations on the basis of two separate criteria. First, morphometry and electron microscopy data showed that E. coli C386 (ompA lpp) cells harboring plasmid vector pEBH21 were rounded while cells of the same strain harboring p4A2HC2 (TpN50+), pWW2200 (OprF+), or pRD87 (OmpA+) were rod shaped. Second, E. coli BRE51 (MC4100 delta sulA-ompA) cells harboring pEBH21 grew poorly at 42 degrees C in minimal medium, while the growth of BRE51 cells harboring p4A2HC2 was similar to that of the parental MC4100 cells. These results demonstrate that the TpN50 protein is functionally equivalent to the E. coli OmpA protein. If TpN50 functions in a similar fashion in T. pallidum, then it may be localized to the treponemal outer membrane.     

Molecular characterization of the pathogen-specific, 34-kilodalton membrane immunogen of Treponema pallidum.

The 34-kilodalton (kDa) antigen of Treponema pallidum subsp. pallidum (T. pallidum) is a pathogen-specific integral membrane protein. DNA sequence analysis of the cloned gene revealed an open reading frame encoding a primary product of 204 residues with a molecular mass of 22,087 daltons. Sequences that correspond to a consensus Escherichia coli promoter and a ribosome-binding site were found upstream from the AUG start codon that begins the open reading frame, suggesting that the cloned gene can use its own regulatory sequences for expression. Examination of the deduced amino acid sequence revealed the presence of a typical procaryotic leader peptide 19 amino acids long; processing results in a mature molecule with a molecular mass of 20,123 daltons. Pulse-chase experiments with E. coli minicells confirmed that the 34-kDa antigen is synthesized as a higher-molecular-weight precursor that is processed to a mature form with the electrophoretic mobility that is characteristic for this protein. The presence in the leader peptide of the sequence Phe-Ser-Ala-Cys suggested that the 34-kDa antigen is a proteolipid. Although hydropathy analysis of the deduced amino acid sequence of the mature 34-kDa antigen predicted that the molecule was primarily hydrophilic, both the native and recombinant 34-kDa molecules displayed hydrophobic biochemical behavior by fractionating into the detergent phase after extraction of intact organisms with Triton X-114. Cell fractionation experiments with E. coli showed that the 34-kDa molecule was localized in both the inner and outer membranes of the recombinant host. The combined data demonstrate that the 34-kDa antigen is an integral membrane protein that behaves in a biochemically consistent manner in both T. pallidum and E. coli.     

Acylation of the 47-kilodalton major membrane immunogen of Treponema pallidum determines its hydrophobicity.

The 47-kilodalton (kDa) major integral membrane immunogen of Treponema pallidum was recently found to be a proteolipid. Similar two-dimensional electrophoretic mobilities and common hydrophobic properties displayed by the native (T. pallidum) and recombinant (Escherichia coli) 47-kDa antigens suggested that the recombinant antigen also possesses covalently bound lipid. Both intact E. coli and E. coli minicells acylated the 47-kDa antigen; immunoprecipitation with a monoclonal antibody specific for the 47-kDa immunogen supported the contention that the acylated product of E. coli corresponds to the cloned T. pallidum antigen. Triton X-114 phase partitioning was used to compare the relative hydrophobicities of 47-kDa molecules synthesized by in vitro and in vivo protein translation systems. The products synthesized by T. pallidum, intact E. coli, or E. coli minicells were hydrophobic, while the protein synthesized in an E. coli cell-free translation system was hydrophilic. Processing experiments with E. coli suggested that the primary gene translation product of the protein is not synthesized in a precursor form, unlike other bacterial proteolipids. These results indicate that the hydrophobicity of the 47-kDa integral membrane protein is conferred substantially by the covalently attached lipid(s). The biochemical similarities between the native and recombinant 47-kDa proteolipids will provide a foundation for future investigations into the structure and immunogenicity of this integral membrane protein of T. pallidum.     

Similarity between the 38-kilodalton lipoprotein of Treponema pallidum and the glucose/galactose-binding (MglB) protein of Escherichia coli.

The recent discovery that abundant and immunogenic lipoproteins constitute the integral membrane proteins of Treponema pallidum has prompted efforts to investigate their importance in the physiology and ultrastructure of the organism and in immune responses during infection. Earlier studies identified a 38-kDa lipoprotein of T. pallidum believed to be specific to the pathogen. In the present study, monoclonal antibodies generated against the 38-kDa lipoprotein of T. pallidum reacted with cognate 37-kDa molecules in the nonpathogens Treponema phagedenis, Treponema denticola, and Treponema refringens. Cloning and expression of the 38-kDa-lipoprotein gene of T. pallidum in Escherichia coli revealed that the recombinant product displayed a slightly larger (39-kDa) apparent molecular mass but remained reactive with anti-38-kDa-protein monoclonal antibodies. The recombinant product was processed and acylated in E. coli. DNA and amino acid sequence analyses indicated an open reading frame encoding 403 amino acids, with the first 25 amino acids corresponding to a leader peptide terminated by a signal peptidase II processing site of Val-Val-Gly-Cys. The predicted mature protein is 378 amino acids in length with a deduced molecular weight of 40,422 (excluding acylation). Southern blotting failed to demonstrate in nonpathogenic treponemes genomic sequences homologous with the 38-kDa-lipoprotein gene of T. pallidum. Computer analysis revealed that the 38-kDa lipoprotein of T. pallidum had 34.2% identity and 58.9% similarity with the glucose/galactose-binding protein (MglB) of E. coli and Salmonella typhimurium. Furthermore, of the 19 amino acids of MglB involved in carbohydrate binding, the 38-kDa lipoprotein had identity with 11. These studies have allowed the first putative functional assignment (carbohydrate binding) to a T. pallidum integral membrane protein. Recognition of this potential physiological role for the 38-kDa lipoprotein underscores the possibility that the membrane biology of T. pallidum may more closely resemble that of gram-positive organisms, which also utilize lipoproteins as anchored transporters, than that of gram-negative bacteria to which T. pallidum often is analogized.     

Monoclonal antibody selection and analysis of a recombinant DNA-derived surface immunogen of Treponema pallidum expressed in Escherichia coli.

Monoclonal antibodies directed against a 34-kilodalton (kDa) surface immunogen of Treponema pallidum were used to select 12 unique T. pallidum DNA-containing Escherichia coli recombinant clones expressing the recombinant form of the 34-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmids in the host cell. Restriction enzyme analyses and Southern hybridization of plasmid DNA demonstrated that all recombinant clones contained common DNA sequences of T. pallidum origin. Further hybridization analyses revealed that the cloned T. pallidum DNA sequences were an accurate representation of the T. pallidum genomic DNA arrangement. Purified immunoglobulin G (IgG) from pooled immune rabbit serum reacted with the clones, while IgG from pooled normal rabbit serum did not. Results of immunological experiments and Southern hybridization indicated that a similar 34-kDa immunogen was present in T. pallidum subsp. pertenue, but it was absent from four species of nonpathogenic treponemes tested, as well as from homogenates of normal rabbit testicular tissue. Metabolic labeling of the E. coli clones with [35S]methionine followed by radioimmunoprecipitation with monoclonal antibodies revealed that the 35S-labeled recombinant and 125I-labeled native (T. pallidum) forms of the antigen had identical electrophoretic mobilities. The production of a complete antigen by E. coli was independent of the orientation of the foreign gene sequence with respect to vector DNA. T. pallidum also produced an apparently identical immunoprecipitable 34-kDa antigen after metabolic labeling with [35S]methionine in the presence of cycloheximide. The apparent specificity of the 34-kDa immunogen for pathogenic treponemes and its native cell surface association on T. pallidum justifies a more intense study of this antigen and its corresponding gene.     

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.     

Expression of Treponema pallidum antigens in Escherichia coli K-12

A colony bank of recombinant plasmids harboring Treponema pallidum DNA inserts has been established in Escherichia coli K-12. By using an in situ immunoassay, we identified four E. coli clones that expressed T. pallidum antigens. Thus, recombinant DNA technology may provide powerful new tools for studying the pathogenesis of T. pallidum infection.     

Serospecificity of a cloned protease-resistant Treponema pallidum--specific antigen expressed in Escherichia coli.

We evaluated the serological reactivity of a protease-resistant antigen designated 4D which was encoded by Treponema pallidum DNA and was expressed in Escherichia coli from recombinant plasmid pAW329. This 19,000-molecular-weight antigen was purified in its native, non-protease-treated form from E. coli sonic extracts by molecular sieving and ion-exchange chromatography. Antibody binding to antigen 4D was detected by a radioimmunoassay. Antigen 4D-specific antibody was detected in 95% of the sera in a Centers for Disease Control syphilis serum panel. It was also detected in 55% of 121 primary syphilis patients, whereas syphilis antibody was detected in 83% of the sera by a fluorescent treponemal antibody absorption test and in 88% of the sera by a T. pallidum microhemagglutination test. In tests of 118 normal sera, less than 3% demonstrated antibody to antigen 4D; these results are similar to microhemagglutination and fluorescent treponemal antibody absorption test results. Rabbit antisera against Treponema phagedenis, Treponema refringens, Treponema denticola, and Treponema vincentii did not react with antigen 4D.     

Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials.     

梅毒螺旋体TP0772蛋白的原核表达和免疫反应性鉴定

目的 利用原核基因工程技术克隆表达梅毒螺旋体TP0772基因,探讨其在梅毒血清学诊断中的应用.方法 PCR扩增获得TP0772基因,构建pET-28b-TP0772重组质粒,转化到大肠杆菌BL21中,以IPTG诱导蛋白质表达,经镍柱纯化后通过质谱技术鉴定.采用免疫印迹法测定其与梅毒患者血清的免疫反应性.建立基于重组TP0772抗原的ELISA间接法并对30份TPPA阳性血清和25份TPP阴性血清进行方法学评价.结果 PCR扩增获得约850 bp的基因片段,成功构建原核表达载体pET-28b-TP0772.目的蛋白分子量约为32kDa,以包涵体的表达形式存在,约占菌体总蛋白的30%.经质谱技术和免疫印迹分析证实重组蛋白为TP0772蛋白,并能够与梅毒患者血清发生特异性结合反应.ELISA测定TPPA阳性血清和阴性血清的符合率分别为93%（28/30）和96%（24/25）.结论 通过DNA重组技术成功获得了重组TP0772蛋白,其与梅毒阳性血清具有良好的免疫反应性,为优化梅毒的血清学诊断方法奠定基础.     

The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein.

Treponema pallidum subsp. pallidum and Escherichia coli incorporated exogenous [3H]palmitate into the 34-kilodalton (kDa) pathogen-specific antigen of T. pallidum. Radiolabeled fatty acid remained associated with the protein upon immunoprecipitation and after boiling in sodium dodecyl sulfate, acetone precipitation, and extensive extractions in organic solvents, suggesting that the fatty acid was covalently bound to the protein. Detection of [3H]palmitate after alkaline and acid hydrolyses confirmed the identity of the incorporated label. Globomycin inhibited maturation of the recombinant 34-kDa antigen, suggesting that E. coli uses the lipoprotein-specific signal peptidase II to process the treponemal antigen. Globomycin also inhibited processing of the 34-kDa antigen, as well as the 44.5- and 15-kDa antigens, in T. pallidum, implying that T. pallidum also possesses the lipoprotein export pathway common to both gram-negative and gram-positive bacteria. Ethanol inhibited processing of the 34-kDa antigen in minicells, suggesting that the 34-kDa antigen normally is translocated through the cytoplasmic membrane. Comparison of the Triton X-114 phase partitioning behavior of the 34-kDa antigen produced either by minicells or by a cell-free translation system indicated that the covalent attachment of fatty acid conferred hydrophobic biochemical properties to the 34-kDa antigen, consistent with the hypothesis that the attached lipid anchors the 34-kDa antigen into the membrane.     

Molecular cloning and characterization of a 35.5-kilodalton lipoprotein of Treponema pallidum.

A clone expressing a 35.5-kDa recombinant treponemal protein was isolated from a genomic DNA library constructed from Treponema pallidum street strain 14. Polyclonal antiserum raised against the recombinant protein reacted with a corresponding native protein of comparable size in T. pallidum that is specific to the pathogenic treponemes. Radiolabeling of the recombinant protein with [3H]palmitate demonstrated that it is lipid modified. Like other recently characterized T. pallidum lipoproteins, the 35.5-kDa lipoprotein partitioned into the detergent phase from T. pallidum cells fractionated with Triton X-114, suggesting that it is an integral membrane protein. Processing of the recombinant 35.5-kDa lipoprotein from a precursor form to a smaller mature form was not evident in pulse-chase experiments. However, pretreatment of Escherichia coli cells expressing the 35.5-kDa lipoprotein with inhibitors of protein processing or translocation revealed the existence of a higher-molecular-mass precursor. Gene fusion studies with the transposon TnphoA demonstrated the presence of an export signal in the 35.5-kDa lipoprotein that promotes the extracytoplasmic localization of a 35.5-kDa lipoprotein-PhoA hybrid.     

Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.     

Purification and characterization of a cloned protease-resistant Treponema pallidum-specific antigen.

A cloned Treponema pallidum antigen, designated 4D, was purified from Escherichia coli predominantly as a 190-kilodalton (kd) polypeptide, although higher oligomeric forms exist. Extensive proteolysis of 4D created a limit digestion product of 90 kd which retained antigenicity with sera from patients with primary, secondary, early latent, late latent, and tertiary syphilis. A molecule indistinguishable from 90-kd 4D in size, isoelectric point, and antigenicity was isolated from T. pallidum after proteolysis. The 190- and 90-kd forms of 4D were stable at 68 degrees C but converted to 19- and 14-kd species, respectively, after boiling in sodium dodecyl sulfate. The low-molecular-weight species did not react with syphilitic sera. Rabbits immunized with the purified 4D antigen developed antibodies which immobilized virulent T. pallidum in a complement-dependent assay system, suggesting that the antigen has a native surface location.