Cultivation of cottontail rabbit epidermal (Sf1Ep) cells on microcarrier beads and their use for suspension cultivation of Treponema pallidum subsp. pallidum

In vitro propagation of Treponema pallidum can be achieved by cocultivation with Sf1Ep cells. This study had two objectives: (i) to achieve suspension cultivation of Sf1Ep cells and (ii) to develop procedures for achieving the replication of T. pallidum in those cell cultures. Seven suspension cultures of Sf1Ep cells yielded an average of 7.2 x 10(8) T. pallidum (36-fold increase) after 12 days.     

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.     

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

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

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.     

Lack of endotoxin in Borrelia hispanica and Treponema pallidum

Borrelia hispanica from infected guinea pigs and Treponema pallidum from testicular syphilomas of rabbits were assayed for the presence of endotoxin with the Limulus lysate test. A suspension of Borrelia, containing 1.3 X 10(8) spirochetes/ml, was nonreactive both when it was tested as intact organisms, and when tested after disruption of the spirochetes by sonication. Eight different suspensions of treponemes, ranging from 0.6 X 10(9) to 3 X 10(9) treponemes/ml, were negative at a 1:10 dilution and were no more active than control suspensions of normal rabbit testes. Therefore, it was concluded that T. pallidum, as well as the Borrelia, possessed no endotoxin.     

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.     

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。     

Genome analysis of Treponema pallidum subsp. pallidum and subsp. pertenue strains: most of the genetic differences are localized in six regions

The genomes of eight treponemes including T. p. pallidum strains (Nichols, SS14, DAL-1 and Mexico A), T. p. pertenue strains (Samoa D, CDC-2 and Gauthier), and the Fribourg-Blanc isolate, were amplified in 133 overlapping amplicons, and the restriction patterns of these fragments were compared. The approximate sizes of the genomes investigated based on this whole genome fingerprinting (WGF) analysis ranged from 1139.3-1140.4 kb, with the estimated genome sequence identity of 99.57-99.98% in the homologous genome regions. Restriction target site analysis, detecting the presence of 1773 individual restriction sites found in the reference Nichols genome, revealed a high genome structure similarity of all strains. The unclassified simian Fribourg-Blanc isolate was more closely related to T. p. pertenue than to T. p. pallidum strains. Most of the genetic differences between T. p. pallidum and T. p. pertenue strains were accumulated in six genomic regions. These genome differences likely contribute to the observed differences in pathogenicity between T. p. pallidum and T. p. pertenue strains. These regions of sequence divergence could be used for the molecular detection and discrimination of syphilis and yaws strains.     

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.     

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.     

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.     

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.     

Treponema pallidum rare outer membrane proteins: analysis of mobility by freeze-fracture electron microscopy.

Freeze-fracture and deep-etch electron microscopy were used to investigate the molecular architecture of the Treponema pallidum outer membrane (OM). Freeze-fracture electron microscopy of treponemes freshly harvested from rabbit testes revealed that the intramembranous particles (IMPs) in both the concave and convex OM leaflets were distributed into alternating areas of relatively high and low particle density; in many OM fractures, IMPs formed rows that ran either parallel to or obliquely across the fracture faces. Statistical analysis (runs test) confirmed that the IMPs were nonrandomly distributed in both OM leaflets. Examination of deep-etched specimens revealed that the particles observed in freeze-fractured OMs also were surface exposed. Combined analysis of deep-etched and cross-fractured treponemes revealed that the OM particles were located in regions of the OM away from the endoflagella and closely apposed to the cytoplasmic membrane-peptidoglycan complex. When treponemes were incubated for extended periods with heat-inactivated immune rabbit syphilitic serum, no alteration in the distribution of OM IMPs was detected. In further experiments, approximately 1:1 mixtures of T. pallidum and Escherichia coli or separate suspensions of the nonpathogenic Treponema phagedenis biotype Reiter were fixed at 34 degrees C or after cooling to 0 degree C (to induce lateral phase separations that would aggregate IMPs). Only particles in the T. pallidum OM failed to aggregate in cells fixed at the lower temperature. The combined data suggest that the mobility of T. pallidum rare OM proteins is limited, perhaps as a result of interactions between their periplasmic domains and components of the peptidoglycan-cytoplasmic membrane complex.     

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.     

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.     

Identification of Treponema pallidum penicillin-binding proteins.

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

Soluble factors from rabbit spleen cells kill and lyse Treponema pallidum in vitro

Antibody and complement immobilize (kill) Treponema pallidum in vitro. Recent evidence also documents immobilization by soluble factors released by activated macrophages and lymphocytes. Immune-mediated lysis of treponemes, however, has not been reported. The findings in this paper focus on apparent treponemal lysis by rabbit splenic cell preparations. Using cells from animals infected testicularly for 9 to 12 days, unfractionated splenic preparations, as well as adherent and nonadherent preparations, killed and lysed T. pallidum. Phagocytosis alone could not explain the detrimental effects of adherent cells. When cytochalasin B was used to block phagocytosis, decreases in treponemal numbers were still detected. In related studies, immune rabbit sera did not enhance treponemicidal activity of the adherent cells. To assess the specificity of these reactions, T. pallidum was incubated with two monocyte-like cell lines (human U937 and mouse P388D1). Neither cell line was detrimental, and treponemal numbers were not lowered. The soluble nature of the treponemicidal factors from adherent and nonadherent preparations was shown by physically separating these cells from the organisms and demonstrating treponemal killing and lysis. In summary, clearance of T. pallidum from infected tissues is probably at least partially attributed to macrophage phagocytosis. Our findings suggest another mechanism involving lytic factors secreted by activated adherent and nonadherent cells.     

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.