Production of tumor necrosis factor alpha by Treponema pallidum, Borrelia burgdorferi s.l., and Leptospira interrogans in isolated rat Kupffer cells

Stimulation of isolated rat Kupffer cells by viable Leptospira interrogans, Treponema pallidum and Borrelia garinii elicited cellular responses resulting in the release of different tumor necrosis factor alpha (TNF-alpha) levels, depending on the spirochetes. L. interrogans induced TNF-alpha levels higher than those achieved with B. garinii and T. pallidum (in this order), but lower than the levels achieved with lipopolysaccharide (LPS). In contrast to L. interrogans, pretreatment of borreliae and treponemes with polymyxin B did not substantially diminish the ability of B. garinii and T. pallidum to stimulate Kupffer cells. Purified T. pallidum lipoproteins TpN47, TmpA, TpN15-TpN17, and B. garinii OspA induced TNF-alpha responses comparable to that achieved by LPS. This response was almost insensitive to the action of polymyxin B.     

Evidence for the presence of lipopolysaccharide in Treponema phagedenis (biotype Reiterii) but not in Treponema pallidum (Nichols)

Abstract Immunoblotting profiles of whole or protease-K-digested organisms with homologous antisera demonstrated the presence of a characteristic ladder pattern of smooth LPS in Treponema phagedenis . Periodic acid silver staining of SDS-PAGE gels confirmed these findings. However, when heterologous or homologous serum was reacted with Treponema pallidum , no such pattern or cross-reactions were observed. The significance of apparent absence of LPS in T. pallidum is discussed.     

Activation of human monocytic cells by Borrelia burgdorferi and Treponema pallidum is facilitated by CD14 and correlates with surface exposure of spirochetal lipoproteins.

Here we examined the involvement of CD14 in monocyte activation by motile Borrelia burgdorferi and Treponema pallidum. B. burgdorferi induced secretion of IL-8 by vitamin D3-matured THP-1 cells, which was inhibited by a CD14-specific mAb known to block cellular activation by LPS and the prototypic spirochetal lipoprotein, outer surface protein A. Enhanced responsiveness to B. burgdorferi also was observed when THP-1 cells were transfected with CD14. Because borreliae within the mammalian host and in vitro-cultivated organisms express different lipoproteins, experiments also were performed with "host-adapted" spirochetes grown within dialysis membrane chambers implanted into the peritoneal cavities of rabbits. Stimulation of THP-1 cells by host-adapted organisms was CD14 dependent and, interestingly, was actually greater than that observed with in vitro-cultivated organisms grown at either 34 degrees C or following temperature shift from 23 degrees C to 37 degrees C. Consistent with previous findings that transfection of Chinese hamster ovary cells with CD14 confers responsiveness to LPS but not to outer surface protein A, B. burgdorferi failed to stimulate CD14-transfected Chinese hamster ovary cells. T. pallidum also activated THP-1 cells in a CD14-dependent manner, although its stimulatory capacity was markedly less than that of B. burgdorferi. Moreover, cell activation by motile T. pallidum was considerably less than that induced by treponemal sonicates. Taken together, these findings support the notion that lipoproteins are the principle component of intact spirochetes responsible for monocyte activation, and they indicate that surface exposure of lipoproteins is an important determinant of a spirochetal pathogen's proinflammatory capacity.     

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.     

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.     

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.     

Complement activation limits the rate of in vitro treponemicidal activity and correlates with antibody-mediated aggregation of Treponema pallidum rare outer membrane protein

A modification of the in vitro immobilization assay together with freeze-fracture analysis was used to determine the factors responsible for the prolonged time required in vitro to achieve killing of Treponema pallidum subsp. pallidum. The modified immobilization assay permitted separate determination of the time required for binding of antibody to the surface of T. pallidum and for C activation. Treponemes were preincubated in heat-inactivated immune rabbit serum (IRS) followed by washing the organisms in 2.5% BSA/PBS to remove unbound IRS antibody before the addition of C. The results showed that a comparable degree of C-dependent killing occurred when treponemes were preincubated in heat-inactivated IRS for either 30 min or 16 h, indicating that treponemicidal antibody rapidly binds to the surface of T. pallidum. Preincubation of treponemes for 17 h in heat-inactivated IRS followed by a 1-h incubation in C resulted in the loss of 80% treponemal motility, indicating that C activation results in rapid killing of T. pallidum. Treponemes preincubated in IRS for 1 h, then incubated for 8 h and 16 h in heat-inactivated normal serum also lost a significant level of motility after the addition of C; in contrast, motility was unaffected after 30 min and 4 h of incubation in heat-inactivated normal serum under similar conditions. These results demonstrate that, whereas antibody binding to and C-mediated killing of treponemes can proceed rapidly, the prolonged time to C activation limits the rate at which treponemicidal activity occurs in vitro. In addition, treponemicidal activity using the modified immobilization assay could not be demonstrated with antiserum against T. pallidum endoflagella, antiserum against proteins solubilized from T. pallidum using the detergent Triton X-114, and a mAb to the T. pallidum r190-kDa "4D" protein, suggesting that these molecules are not accessible to surface binding antibody. Freeze-fracture analysis, recently used in our laboratory to demonstrate that the outer membrane of T. pallidum has rare constituent protein, was utilized to demonstrate outer membrane target Ag of IRS antibody. T. pallidum rare outer membrane protein (TROMP) molecules were shown in freeze-fracture electron micrographs to be consistently aggregated following a 16-h incubation of treponemes in IRS. In contrast, no aggregation of TROMP was present in treponemes incubated in normal rabbit serum for 16 h or in treponemes incubated in IRS for 2 h. These findings suggest that the rate of C activation leading to in vitro treponemicidal activity is limited by the time required for aggregation of antibody-bound TROMP molecules.     

Characterization of the serum requirement for macrophage-mediated killing of Treponema pallidum ssp. pallidum: Relationship to the development of opsonizing antibodies

Abstract Serum pools were collected from rabbits bled at various times after intra-testicular infection with Treponema pallidum ssp. pallidum . These were tested for their ability to opsonize T. pallidum and promote killing of the organisms by macrophages. Compared to normal sera, significant opsonization was first seen on day 10 of infection as measured by both ingestion ( P < 0.001) and macrophage-mediated killing ( P = 0.006); significant levels of functional antibodies persisted through 300 days of infection. Although opsonic activity peaked early in infection, antibodies that promoted optimal macrophage-mediated killing developed much later, suggesting that these two functions may represent activities of antibodies with differing specificities or affinities. The initial development of antibodies that augment both phagocytosis and killing corresponds with the in vivo clearance of treponemes from the primary site of infection. These observations support the hypothesis that macrophages are the major effector mechanism for elimination of T. pallidum during early syphilis infection.     

Assessment of cell-surface exposure and vaccinogenic potentials of Treponema pallidum candidate outer membrane proteins

Syphilis, a sexually transmitted infection caused by the spirochetal bacterium Treponema pallidum, remains a global public health problem. T. pallidum is believed to be an extracellular pathogen and, as such, the identification of T. pallidum outer membrane proteins that could serve as targets for opsonic or bactericidal antibodies has remained a high research priority for vaccine development. However, the identification of T. pallidum outer membrane proteins has remained highly elusive. Recent studies and bioinformatics have implicated four treponemal proteins as potential outer membrane proteins (TP0155, TP0326, TP0483 and TP0956). Indirect immunofluorescence assays performed on treponemes encapsulated within agarose gel microdroplets failed to provide evidence that any of these four molecules were surface-exposed in T. pallidum. Second, recombinant fusion proteins corresponding to all four candidate outer membrane proteins were used separately, or in combination, to vaccinate New Zealand White rabbits. Despite achieving high titers (>1:50,000) of serum antibodies, none of the rabbits displayed chancre immunity after intradermal challenge with viable T. pallidum.     

Surface properties of Treponema pallidum in relation to phagocytosis by human polymorphonuclear leucocytes in vitro

Surface charge and hydrophobicity of Treponema pallidum have been investigated in relation to phagocytosis by human polymorphonuclear leucocytes (PMNs) in vitro. The treponemal surface was relatively hydrophobic and negatively charged but despite these properties, phagocytosis, as assessed by luminol-enhanced chemiluminescence, was minimal in the absence of serum. Preopsonization of bacteria with serum reduced surface hydrophobicity but promoted phagocytosis, suggesting that specific immune mechanisms may be more important in controlling phagocytosis of T. pallidum in vitro than non-specific surface properties. T. pallidum evoked a much weaker chemiluminescence response from PMNs than the non-pathogenic treponeme Treponema phagedenis biotype Reiterii even though similar numbers of bacteria were phagocytosed, suggesting differences in the reactivity of the surface components of the two organisms toward PMNs. The reactivity of T. pallidum towards PMNs could be increased by removal of the bacterial outer membrane by Triton X-100 treatment. These observations reinforce the suggestion that the outer surface of T. pallidum is inherently inert.     

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.     

Cloning and expression of treponema pallidum common antigen (Tp-4) in Escherichia coli K12

A library of Treponema pallidum DNA was constructed using a cosmid cloning system. Sixteen hundred Escherichia coli recombinant clones were generated covering the T. pallidum genome with a probability of 99%. Three hundred of the clones were screened for expression of T. pallidum antigens by a modified rocket immunoelectrophoresis technique using a polyspecific antiserum to T. pallidum. One clone was identified which produced the 'common antigen' (CA) of T. pallidum (Tp-4). CA shares epitopes with antigens present in more than 50 different bacterial species, but nothing is known about its structure, function and localization. The recombinant E. coli clone will be of value for a structural analysis of the CA gene.     

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.     

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.     

Selective release of the Treponema pallidum outer membrane and associated polypeptides with Triton X-114.

The effects of the nonionic detergent Triton X-114 on the ultrastructure of Treponema pallidum subsp. pallidum are presented in this study. Treatment of Percoll-purified motile T. pallidum with a 1% concentration of Triton X-114 resulted in cell surface blebbing followed by lysis of blebs and a decrease in diameter from 0.25-0.35 micron to 0.1-0.15 micron. Examination of thin sections of untreated Percoll-purified T. pallidum showed integrity of outer and cytoplasmic membranes. In contrast, thin sections of Triton X-114-treated treponemes showed integrity of the cytoplasmic membrane but loss of the outer membrane. The cytoplasmic cylinders generated by detergent treatment retained their periplasmic flagella, as judged by electron microscopy and immunoblotting. Recently identified T. pallidum penicillin-binding proteins also remained associated with the cytoplasmic cylinders. Proteins released by Triton X-114 at 4 degrees C were divided into aqueous and hydrophobic phases after incubation at 37 degrees C. The hydrophobic phase had major polypeptide constituents of 57, 47, 38, 33-35, 23, 16, and 14 kilodaltons (kDa) which were reactive with syphilitic serum. The 47-kDa polypeptide was reactive with a monoclonal antibody which has been previously shown to identify a surface-associated T. pallidum antigen. The aqueous phase contained the 190-kDa ordered ring molecule, 4D, which has been associated with the surface of the organisms. Full release of the 47- and 190-kDa molecules was dependent on the presence of a reducing agent. These results indicate that 1% Triton X-114 selectively solubilizes the T. pallidum outer membrane and associated proteins of likely outer membrane location.     

Autoantibodies to creatine kinase in rabbits infected with Treponema pallidum

Sera from rabbits infected intratesticularly with Treponema pallidum (Nichols) for 30 days were examined for autoantibody reactivity against muscle and testis extracts by Western immunoblotting. Syphilitic sera (30 day) reacted with an autoantigen of 43,000 daltons in muscle extracts. The antigen was shown to be creatine kinase (CK). Studies with the use of an anti-CK ELISA showed that the autoantibody to CK first appeared 3 wk after infection, declined by 7 wk infection, and was absent in rabbits "mock"-infected with heat-killed T. pallidum. CK activity was not detected in sonicated or intact, washed T. pallidum, suggesting that the antibody was not produced in response to treponemal CK.     

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.     

Ultrastructure of Lipopolysaccharide Isolated from Treponema pallidum

A lipopolysaccharide (LPS) fraction was extracted from Nichols, nonpathogenic Treponema pallidum by the hot, phenol-water procedure. The LPS was freed of nucleic acids and water-soluble proteins by successive exposures to ribonuclease, deoxyribonuclease, and Pronase. Purified LPS responded positively in a colorimetric assay for lipopolysaccharide. Electron microscope examination of the LPS both before and after purification demonstrated a heterogeneous mixture of forms including spheres, doughnuts, and ribbons. The trilaminar nature of the ribbon forms was observed by both negative staining and thin sectioning. Lyophilization of the LPS caused an increase in the number and length of ribbon forms seen. Results suggest that the surface layers of treponemes are similar to those of gram-negative bacteria.     

Further evidence for hyaluronidase activity of Treponema pallidum

The presence of hyaluronidase in preparations of Treponema pallidum was previously shown using acidified bovine serum albumin reactions and Ouchterlony immunodiffusion. To expand on these preliminary findings more sensitive techniques of viscometry, additional immunologic reactions, and altered capillary permeability were used to characterize treponemal-associated hyaluronidase. The pathogens T. pallidum and T. pertenue degraded hyaluronic acid, whereas the nonpathogens T. denticola and T. vincentii did not. As syphilitic infection progressed, hyaluronidase activity decreased; organisms harvested from 14-day testicular infections degraded hyaluronic acid less rapidly than organisms from 4-day infections. Uninfected rabbit testicular extract also exhibited significant enzyme activity. The neutralizing activity of immune sera was decreased by prior adsorption with bovine hyaluronidase, suggesting that some of the neutralizing factors are associated with this enzyme. Radioimmunoassay was used to quantitate antibodies to hyaluronidase in immune sera. Antihyaluronidase sera were isolated from rabbits immunized with bovine hyaluronidase. Treponema pallidum, as well as uninfected rabbit testicular extract, cross-reacted with these antisera. Immunofluorescence indicated that the hyaluronidase was uniformly distributed along the treponemal surface. As a final indicator of hyaluronidase activity, alterations in capillary permeability were detected 1 h after intradermal injection of T. pallidum.