Passage of Treponema pallidum Through Membrane Filters of Various Pore Sizes

The passage of Treponema pallidum through commercially available Millipore membrane filters of various pore sizes was examined. No microscopically detectable organisms passed through a filter with a pore diameter of 0.22 mum. As pore size was increased, progressively more organisms passed through. Motile organisms passed through filters to a greater extent than nonmotile ones; however, 22% of the motile and 50% of the nonmotile T. pallidum organisms did not pass through the largest pore diameter tested (14.0 mum). Filtration of T. pallidum suspensions through membrane filters may offer a way of separating the organisms from larger particles of debris which accompany their extraction from rabbit testicular syphilomas.     

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.     

Antigenic complexity of Treponema pallidum: antigenicity and surface localization of major polypeptides

The protein structure of Treponema pallidum was characterized by two-dimensional electrophoresis (2DE), consisting of isoelectric focusing (IEF, pH 5 to 7) in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. Up to 85 major polypeptide species could be detected in the organisms in 2DE gels by Coomassie Blue staining. The antigenicity of the individual polypeptides was determined by transferring the 2DE pattern to nitrocellulose paper and utilizing a sensitive immunoperoxidase procedure to demonstrate the reactivity of immunoglobulins in sera obtained from rabbits infected intratesticularly at least 6 mo previously. The infected rabbit serum reacted with virtually every major polypeptide detectable by protein staining techniques, indicating that infected rabbits produce antibodies against nearly all major T. pallidum proteins at the time when the animals exhibit systemic resistance to reinfection. Surface radioiodination of freshly purified T. pallidum by an Iodogen procedure yielded preferential labeling of a major polypeptide with an apparent m.w. of 39,000. The results of this study indicate that the antigenic complexity of T. pallidum is much greater than described previously. The 39-kd polypeptide appears to be a major surface constituent of T. pallidum and as such may play an important role in the induction of immunity to syphilis.     

The role of respiratory protection on increased survival of Treponema pallidum (Nichols) when cocultivated with mammalian cells in vitro

The ability of mammalian cells in tissue culture to protect against oxygen toxicity for Treponema pallidum was examined. Addition of catalase to the incubation medium enhanced T. pallidum survival when co-incubation was carried out under aerobic conditions. When co-incubation was carried out under 3% oxygen, catalase had no enhancing effect on survival despite the fact it was still highly stimulatory when T. pallidum was incubated under 3% oxygen in the same medium with no tissue culture cells present. Inactivation of the catalase present endogenously in the mammalian cells by the addition of the catalase inhibitor 3-amino-1,2,4-triazole largely eliminated the enhancing effect of mammalian cells on the survival of T. pallidum under 3% oxygen. Increasing the oxygen consumption of the host mammalian cells with 0.1 mM 2,4-dinitrophenol enhanced T. pallidum under both aerobic and microaerobic conditions; a much greater effect was seen under aerobic conditions. The results indicated that mammalian cells offer significant protection against toxic oxygen reduction products for T. pallidum in vitro under microaerobic conditions.     

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.     

Detection and functional characterization of early appearing antibodies in rabbits with experimental syphilis

Following testicular infection of rabbits with Treponema pallidum, different antibodies become detectable initially at the time of healing. Experiments were performed to determine a functional role for these antibodies. Rabbits were sacrificed after 4-8 days. Treponemal numbers steadily increased for 10-12 days. Thereafter, host defenses were sufficiently stimulated to begin clearing the organisms. Antibodies in serum and antibodies localized at the site of infection were quantitated using radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) techniques. Anti-treponemal IgG was detected as early as day 4. Quantities of antibody correspondingly increased with time following infection. Treponema pallidum was harvested 7 and 14 days postinfection and tested for surface antibodies. With increasing days postinfection, more antibody was found on the organisms. Two functional properties of these antibodies were shown. Sera from 24 of 45 rabbits infected for 14 days immobilized T. pallidum in the presence of complement and 14-day sera blocked the attachment of T. pallidum to tissue culture cells. We suggest that antibody-mediated, complement-dependent immobilization of T. pallidum and blockage of attachment are at least partially responsible for healing of testicular lesions.     

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.     

Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D.

Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a protease-resistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.     

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.     

Biology of Treponema pallidum: correlation of functional activities with genome sequence data

Aspects of the biology of T. pallidum subsp. pallidum, the agent of syphilis, are examined in the context of a century of experimental studies and the recently determined genome sequence. T. pallidum and a group of closely related pathogenic spirochetes have evolved to become highly invasive, persistent pathogens with little toxigenic activity and an inability to survive outside the mammalian host. Analysis of the genome sequence confirms morphologic studies indicating the lack of lipopolysaccharide and lipid biosynthesis mechanisms, as well as a paucity of outer membrane protein candidates. The metabolic capabilities and adaptability of T. pallidum are minimal, and this relative deficiency is reflected by the absence of many pathways, including the tricarboxylic acid cycle, components of oxidative phosphorylation, and most biosynthetic pathways. Although multiplication of T. pallidum has been obtained in a tissue culture system, continuous in vitro culture has not been achieved. The balance of oxygen utilization and toxicity is key to the survival and growth of T. pallidum, and the genome sequence reveals a similarity to lactic acid bacteria that may be useful in understanding this relationship. The identification of relatively few genes potentially involved in pathogenesis reflects our lack of understanding of invasive pathogens relative to toxigenic organisms. The genome sequence will provide useful raw data for additional functional studies on the structure, metabolism, and pathogenesis of this enigmatic organism.     

Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum.

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.     

Effects of molecular oxygen, oxidation-reduction potential, and antioxidants upon in vitro replication of Treponema pallidum subsp. pallidum

The effects of various concentrations of dithiothreitol, molecular oxygen, and several antioxidants upon the in vitro replication of Treponema pallidum were studied. The optimal dithiothreitol concentration was between 0.65 and 1.62 mM, and the optimum oxygen concentration was 3.0% +/- 0.5% in both the presence and absence of additional antioxidants. It was discovered that the reduced sulfhydryl concentration and the oxidation-reduction potential of the medium were stabilized after 5 days. The water-soluble antioxidants cobalt chloride, cocarboxylase, mannitol, and histidine were individually tested for their ability to increase treponemal growth in vitro. The optimum concentrations for these antioxidants were 21 nM, 4.3 nM, 0.55 mM, and 0.23 mM, respectively. When combined at these concentrations, the mixture of antioxidants stimulated the in vitro replication of T. pallidum. The number of treponemes in cultures with the antioxidants averaged a 59-fold increase, compared with a 43-fold increase in cultures lacking the antioxidants. It was further demonstrated that histidine and mannitol were the most critical components of this mixture. Catalase and superoxide dismutase were investigated for their ability to promote the growth and maintain viability of T. pallidum in tissue culture. The optimum concentrations for these enzymes were 10,000 U/liter and 25,000 U/liter, respectively. When these enzymes and the above antioxidants were combined and added to a chemically reduced modified Eagle medium, the treponemes increased an average of 70-fold, compared with an average of 35-fold in cultures lacking them. Furthermore, this medium, T. pallidum culture medium, supported the replication of T. pallidum at oxygen concentrations from 5 to 7% with little loss in yield or viability. The lipid-soluble antioxidants vitamin A and vitamin E acetate were also shown to enhance the in vitro growth of T. pallidum in this medium.     

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.     

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.     

TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein

Definitive identification of Treponema pallidum rare outer membrane proteins (OMPs) has long eluded researchers. TP0326, the sole protein in T. pallidum with sequence homology to a Gram-negative OMP, belongs to the BamA family of proteins essential for OM biogenesis. Structural modelling predicted that five polypeptide transport-associated (POTRA) domains comprise the N-terminus of TP0326, while the C-terminus forms an 18-stranded amphipathic β-barrel. Circular dichroism, heat modifiability by SDS-PAGE, Triton X-114 phase partitioning and liposome incorporation supported these topological predictions and confirmed that the β-barrel is responsible for the native protein's amphiphilicity. Expression analyses revealed that native TP0326 is expressed at low abundance, while a protease-surface accessibility assay confirmed surface exposure. Size-exclusion chromatography and blue native polyacrylamide gel electrophoresis revealed a modular Bam complex in T. pallidum larger than that of Escherichia coli. Non-orthologous ancillary factors and self-association of TP0326 via its β-barrel may both contribute to the Bam complex. T. pallidum-infected rabbits mount a vigorous antibody response to both POTRA and β-barrel portions of TP0326, whereas humans with secondary syphilis respond predominantly to POTRA. The syphilis spirochaete appears to have devised a stratagem for harnessing the Bam pathway while satisfying its need to limit surface antigenicity.     

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.     

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.     

梅毒螺旋体膜蛋白研究进展

梅毒螺旋体苍白亚种（ Tp）引起的梅毒是一种严重危害人类健康的慢性性传播性疾病,其发病具有多阶段、进行性的特点。由于Tp尚不能体外培养,抗原获取困难,其致病机制研究尚不清楚。随着Tp全基因序列的解析,重组Tp膜蛋白的成功表达及蛋白功能结构日益明确,为Tp发病机制的研究及疫苗的研制奠定了良好的基础。对于Tp主要外膜蛋白的结构、功能研究进展进行了综述。     

Correlation of treponemicidal activity in normal human serum with the presence of IgG antibody directed against polypeptides of Treponema phagedenis biotype Reiter and Treponema pallidum, Nichols strain

Normal human serum (NHS) was shown to have complement-dependent treponemicidal activity against both Treponema pallidum and Treponema phagedenis biotype Reiter (TPR) by employing in vitro-in vivo neutralization and TPR plaque assays, respectively. The molecular basis of NHS treponemicidal activity was studied by immunoblot analysis in conjunction with treponemicidal assays. Five major T. pallidum polypeptide bands (47kDa, 35kDa, 33kDa doublet, and 30 kDa) and three major TPR polypeptide bands (47kDa and 33kDa doublet) bound IgG present in NHS. Absorption of NHS with TPR completely removed both TPR and T. pallidum treponemicidal activity; corresponding immunoblots demonstrated a significant removal of IgG antibody against all three TPR polypeptide bands as well as four T. pallidum polypeptide bands (30kDa, 33kDa doublet, and 35kDa). In contrast, T. pallidum absorption of NHS was found to remove treponemicidal activity against T. pallidum but not TPR; corresponding Western blots showed the complete removal of IgG antibody against all but one T. pallidum polypeptide band (47kDa) but no detectable loss in IgG antibody against the TPR polypeptides. These results suggest that antibody in NHS generated against nonpathogenic, indigenous treponemes is responsible for the T. pallidum treponemicidal activity. Furthermore, the treponemicidal activity against T. pallidum correlated with the presence of IgG antibody against T. pallidum polypeptides of 30kDa, 35kDa, and a 33kDa doublet.     

Fatty acids of Treponema pallidum and Borrelia burgdorferi lipoproteins.

A fundamental ultrastructural feature shared by the spirochetal pathogens Treponema pallidum subsp. pallidum (T. pallidum) and Borrelia burgdorferi, the etiological agents of venereal syphilis and Lyme disease, respectively, is that their most abundant membrane proteins contain covalently attached fatty acids. In this study, we identified the fatty acids covalently bound to lipoproteins of B. burgdorferi and T. pallidum and examined potential acyl donors to these molecules. Palmitate was the predominant fatty acid of both B. burgdorferi and T. pallidum lipoproteins. T. pallidum lipoproteins also contained substantial amounts of stearate, a fatty acid not typically prevalent in prokaryotic lipoproteins. In both spirochetes, the fatty acids of cellular lipids differed from those of their respective lipoproteins. To characterize phospholipids in these organisms, spirochetes were metabolically labeled with [3H]palmitate or [3H]oleate; B. burgdorferi contained only phosphatidylglycerol and phosphatidylcholine, while T. pallidum contained phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and cardiolipin. Although palmitate predominated in the lipoproteins, there were no apparent differences in the incorporation of these two fatty acids into phospholipids (putative acyl donors). Phospholipase A1 and A2 digestion of phosphatidylcholine from B. burgdorferi and T. pallidum labeled with either [3H]palmitate or [3H]oleate also revealed that neither fatty acid was incorporated preferentially into the 1 and 2 positions (potential acyl donor sites) of the glycerol backbone. The combined findings suggest that fatty acid utilization during lipoprotein synthesis is determined largely by the fatty acid specificities of the lipoprotein acyl transferases. These findings also provide the basis for ongoing efforts to elucidate the relationship between lipoprotein acylation and the physiological functions and inflammatory activities of these molecules.