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PubMed2000

Neelaredoxin, an iron-binding protein from the syphilis spirochete, Treponema pallidum, is a superoxide reductase.

Treponema pallidum, the causative agent of venereal syphilis, is a microaerophilic obligate pathogen of humans. As it disseminates hematogenously and invades a wide range of tissues, T. pallidum presumably must tolerate substantial oxidative stress. Analysis of the T. pallidum genome indicates that the syphilis spirochete lacks most of the iron-binding proteins present in many other bacterial pathogens, including the oxidative defense enzymes superoxide dismutase, catalase, and peroxidase, but does possess an orthologue (TP0823) for neelaredoxin, an enzyme of hyperthermophilic and sulfate-reducing anaerobes shown to possess superoxide reductase activity. To analyze the potential role of neelaredoxin in treponemal oxidative defense, we examined the biochemical, spectroscopic, and antioxidant properties of recombinant T. pallidum neelaredoxin. Neelaredoxin was shown to be expressed in T. pallidum by reverse transcriptase-polymerase chain reaction and Western blot analysis. Recombinant neelaredoxin is a 26-kDa alpha(2) homodimer containing, on average, 0.7 iron atoms/subunit. Mössbauer and EPR analysis of the purified protein indicates that the iron atom exists as a mononuclear center in a mixture of high spin ferrous and ferric oxidation states. The fully oxidized form, obtained by the addition of K(3)(Fe(CN)(6)), exhibits an optical spectrum with absorbances at 280, 320, and 656 nm; the last feature is responsible for the protein's blue color, which disappears upon ascorbate reduction. The fully oxidized protein has a A(280)/A(656) ratio of 10.3. Enzymatic studies revealed that T. pallidum neelaredoxin is able to catalyze a redox equilibrium between superoxide and hydrogen peroxide, a result consistent with it being a superoxide reductase. This finding, the first description of a T. pallidum iron-binding protein, indicates that the syphilis spirochete copes with oxidative stress via a primitive mechanism, which, thus far, has not been described in pathogenic bacteria.

Amino Acid Sequence↗
PubMed2000

Superoxide reductase as a unique defense system against superoxide stress in the microaerophile Treponema pallidum.

Aerobic life requires the presence of antioxidant enzymes, such as superoxide dismutase, catalase, and peroxidase to eliminate deleterious oxygen derivatives. Treponema pallidum, a microaerophilic bacterium responsible for venereal syphilis, is an interesting organism because it lacks all of the above-mentioned enzymes, as deduced from its recently sequenced genome. In this paper, we describe a gene in T. pallidum with sequence homologies to a new class of antioxidant systems, named superoxide reductases, recently isolated from sulfate-reducing bacteria (Lombard, M., Fontecave, M., Touati, D., and Nivière, V. (2000) J. Biol. Chem. 275, 115-121). We report that (i) expression of the T. pallidum gene fully restored to a superoxide dismutase-deficient Escherichia coli mutant the ability to grow under aerobic conditions; (ii) the corresponding protein displays a strong superoxide reductase activity; and (iii) the T. pallidum protein contains only one mononuclear nonheme ferrous center, able to reduce superoxide selectively and efficiently, whereas previously characterized superoxide reductase from Desulfoarculus baarsii contains an additional rubredoxin-like ferric center. These results suggest that T. pallidum antioxidant defenses rely on a new class of superoxide reductase and raise the question of the importance of superoxide reductases in mechanisms for detoxifying superoxide radicals.

Amino Acid Sequence↗
PubMed2002

Crystal structure of the 47-kDa lipoprotein of Treponema pallidum reveals a novel penicillin-binding protein.

Syphilis is a complex sexually transmitted disease caused by the spirochetal bacterium Treponema pallidum. T. pallidum has remained exquisitely sensitive to penicillin, but the mode of action and lethal targets for beta-lactams are still unknown. We previously identified the T. pallidum 47-kDa lipoprotein (Tp47) as a penicillin-binding protein (PBP). Tp47 contains three hypothetical consensus motifs (SVTK, TEN, and KTG) that typically form the active center of other PBPs. Yet, in this study, mutations of key amino acids within these motifs failed to abolish the penicillin binding activity of Tp47. The crystal structure of Tp47 at a resolution of 1.95 A revealed a fold different from any other known PBP; Tp47 is predominantly beta-sheet, in contrast to the alpha/beta-fold common to other PBPs. It comprises four distinct domains: two complex beta-sheet-containing N-terminal domains and two C-terminal domains that adopt immunoglobulin-like folds. The three hypothetical PBP signature motifs do not come together to form a typical PBP active site. Furthermore, Tp47 is unusual in that it displays beta-lactamase activity (k(cat) for penicillin = 271 +/- 6 s(-1)), a feature that hindered attempts to identify the active site in Tp47 by co-crystallization and mass spectrometric techniques. Taken together, Tp47 does not fit the classical structural and mechanistic paradigms for PBPs, and thus Tp47 appears to represent a new class of PBP.

Acylation↗
PubMed2003

The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase.

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E. coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm. The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo. Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T. pallidum encounters high Zn2+ levels.

ATP-Binding Cassette Transporters↗
PubMed2003

Lysine 238 is an essential residue for alpha,beta-elimination catalyzed by Treponema denticola cystalysin.

Treponema denticola cystalysin is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the alpha,beta-elimination of l-cysteine to pyruvate, ammonia, and H2S. Similar to other PLP enzymes, an active site Lys residue (Lys-238) forms an internal Schiff base with PLP. The mechanistic role of this residue has been studied by an analysis of the mutant enzymes in which Lys-238 has been replaced by Ala (K238A) and Arg (K238R). Both apomutants reconstituted with PLP bind noncovalently approximately 50% of the normal complement of the cofactor and have a lower affinity for the coenzyme than that of wild-type. Kinetic analyses of the reactions of K238A and K238R mutants with glycine compared with that of wild-type demonstrate the decrease of the rate of Schiff base formation by 103- and 7.5 x 104-fold, respectively, and, to a lesser extent, a decrease of the rate of Schiff base hydrolysis. Thus, a role of Lys-238 is to facilitate formation of external aldimine by transimination. Kinetic data reveal that the K238A mutant is inactive in the alpha,beta-elimination of l-cysteine and beta-chloro-l-alanine, whereas K238R retains 0.3% of the wild-type activity. These data, together with those derived from a spectral analysis of the reaction of Lys-238 mutants with unproductive substrate analogues, indicate that Lys-238 is an essential catalytic residue, possibly participating as a general base abstracting the Calpha-proton from the substrate and possibly as a general acid protonating the beta-leaving group.

Catalysis↗
PubMed2004

Site-directed mutagenesis provides insight into racemization and transamination of alanine catalyzed by Treponema denticola cystalysin.

In addition to alpha, beta-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at Calpha/C4' of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys --> Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k(cat)/K(m) is reduced 3.5-fold for alpha, beta-elimination, whereas it is reduced 300-400-fold for racemization. Y123F has approximately 18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.

Alanine↗
PubMed2004

Structural evidence that the 32-kilodalton lipoprotein (Tp32) of Treponema pallidum is an L-methionine-binding protein.

A structure-to-function approach was undertaken to gain insights into the potential function of the 32-kDa membrane lipoprotein (Tp32) of Treponema pallidum, the syphilis bacterium. The crystal structure of rTp32 (determined at a resolution of 1.85 A) shows that the organization of rTp32 is similar to other periplasmic ligand-binding proteins (PLBPs), in that it consists of two alpha/beta domains, linked by two crossovers, with a binding pocket between them. In the pocket, a molecule of L-methionine was detected in the electron density map. Residues from both domains interact with the ligand. One of the crossover regions is comprised of a 3(10)-helix, a feature not typical in other ligand-binding proteins. Sequence comparison shows strong similarity to other hypothetical methionine-binding proteins. Together, the data support the notion that rTp32 is a component of a periplasmic methionine uptake transporter system in T. pallidum.

Apolipoproteins↗
PubMed2006

The PnrA (Tp0319; TmpC) lipoprotein represents a new family of bacterial purine nucleoside receptor encoded within an ATP-binding cassette (ABC)-like operon in Treponema pallidum.

Treponema pallidum, the bacterial agent of syphilis, cannot be cultivated in vitro. This constraint has severely impeded the study of the membrane biology of this complex human pathogen. A structure-to-function approach thus was adopted as a means of discerning the likely function of Tp0319, a 35-kDa cytoplasmic membrane-associated lipoprotein of T. pallidum formerly designated as TmpC. A 1.7-A crystal structure showed that recombinant Tp0319 (rTp0319) consists of two alpha/beta domains, linked by three crossovers, with a deep cleft between them akin to ATP-binding cassette (ABC) receptors. In the cleft, a molecule of inosine was bound. Isothermal titration calorimetry demonstrated that rTp0319 specifically binds purine nucleosides (dissociation constant (Kd) approximately 10(-7) M). This predilection for purine nucleosides by rTp0319 is consistent with its likely role as a receptor component of a cytoplasmic membrane-associated transporter system. Reverse transcription-PCR analysis of RNA isolated from rabbit tissue-extracted T. pallidum additionally showed that tp0319 is transcriptionally linked to four other downstream open reading frames, thereby supporting the existence of an ABC-like operon (tp0319-0323). We herein thus re-name tp0319 as purine nucleoside receptor A (pnrA), with its operonic partners tp0320-0323 designated as pnrB-E, respectively. Our study not only infers that PnrA transports purine nucleosides essential for the survival of T. pallidum within its obligate human host, but to our knowledge, this is the first description of an ABC-type nucleoside transport system in any bacterium. PnrA has been grouped with a functionally uncharacterized protein family (HBG016869), thereby implying that other members of the family may have similar nucleoside-binding function(s).

Adenosine Triphosphate↗
PubMed2006

Crystal structure of the Tp34 (TP0971) lipoprotein of treponema pallidum: implications of its metal-bound state and affinity for human lactoferrin.

The Tp34 (TP0971) membrane lipoprotein of Treponema pallidum, an obligate human pathogen and the agent of syphilis, was previously reported to have lactoferrin binding properties. Given the non-cultivatable nature of T. pallidum, a structure-to-function approach was pursued to clarify further potential relationships between the Tp34 structural and biochemical properties and its propensity to bind human lactoferrin. The crystal structure of a nonacylated, recombinant form of Tp34 (rTp34), solved to a resolution of 1.9A(,) revealed two metaloccupied binding sites within a dimer; the identity of the ion most likely was zinc. Residues from both of the monomers contributed to the interfacial metal-binding sites; a novel feature was that the delta-sulfur of methionine coordinated the zinc ion. Analytical ultracentrifugation showed that, in solution, rTp34 formed a metal-stabilized dimer and that rTp34 bound human lactoferrin with a stoichiometry of 2:1. Isothermal titration calorimetry further revealed that rTp34 bound human lactoferrin at high (submicromolar) affinity. Finally, membrane topology studies revealed that native Tp34 is not located on the outer surface (outer membrane) of T. pallidum but, rather, is periplasmic. How propensity of Tp34 to bind zinc and the iron-sequestering lactoferrin may relate overall to the biology of T. pallidum infection in humans is discussed.

Crystallography, X-Ray↗
PubMed2002

Treponema pallidum induces up-regulation of interstitial collagenase in human dermal fibroblasts.

In this study we investigated the capability of Treponema pallidum to stimulate human dermal fibroblasts to produce interstitial collagenase (MMP-1), which is needed to degrade type I collagen, the most abundant component of the human dermis. When T. pallidum was added to human dermal fibroblast culture, both the amount of secreted MMP-1 and its mRNA levels were increased. Our results show that T. pallidum can stimulate host human fibroblasts to increase the synthesis of MMP-1, which may act as a virulence factor of the organism.

Blotting, Northern↗
PubMed1998

Passive particle agglutination test for screening of Treponema Pallidum antibodies in blood bank routine.

In most laboratories and blood banks, the TP.PA test has replaced the TPHA test for the screening of Treponema pallidum antibodies. In this study we show that the 1:20 serum dilution should be used to discriminate between negative and positive samples instead of the recommended 1:40 dilution, thus increasing sensitivity without a significant loss of specificity.

Antibodies, Bacterial↗
PubMed1985

Fibronectin mediates Treponema pallidum cytadherence through recognition of fibronectin cell-binding domain.

The specificity of the interaction between Treponema pallidum and fibronectin was demonstrated. Treatment of host cells with only antifibronectin sera and not anticollagen or antilaminin sera, inhibited treponemal cytadsorption. Incubation of fibronectin-coated coverslips with monoclonal antibody to the cell-binding domain of fibronectin reduced treponemal attachment to the same extent as antifibronectin serum. Both iodinated fibronectin and iodinated cell-binding domain bound to T. pallidum in a saturable manner. Specificity of the T. pallidum association with the cell-binding domain was the most effective inhibitor of the binding of either radioiodinated cell-binding domain or fibronectin to T. pallidum. Scatchard analysis gave Kd on the order of 10(-7) M for both cell-binding domain and fibronectin binding to T. pallidum, consistent with the high affinity interaction of these organisms with host cell surfaces. Finally, the same level of attachment of treponemes was achieved on coverslips coated with cell-binding domain as that observed for organisms incubated with fibronectin, indicating that the cell-binding domain polypeptide is functionally identical to fibronectin in mediating T. pallidum adherence.

Adhesiveness↗
PubMed1986

Isolation of a Treponema pallidum gene encoding immunodominant outer envelope protein P6, which reacts with sera from patients at different stages of syphilis.

A phage directing the synthesis of an abundant 45-kD Treponema pallidum surface protein was isolated from an EMBL-4 bacteriophage lambda library of T. pallidum DNA. The recombinant phage was identified using an mAb that was directed toward an immunodominant, outer envelope T. pallidum protein designated P6. The recombinant P6 protein possessed the same mol mass as the native treponemal antigen detected from total T. pallidum protein preparations, confirming the cloning of the structural gene for this molecule. Furthermore, E. coli was transformed by a 4.5-kb Eco RI lambda insert fragment subcloned into the plasmid vector pUC19. These transformed cells expressed and translocated the 45-kD protein to their outer membranes. Finally, all sera from patients with different stages of syphilis (primary, secondary, and latent) contained antibody reactive to this protein.

Bacterial Outer Membrane Proteins↗
PubMed1999

Treponema pallidum major sheath protein homologue Tpr K is a target of opsonic antibody and the protective immune response.

We have identified a family of genes that code for targets for opsonic antibody and protective immunity in T. pallidum subspecies pallidum using two different approaches, subtraction hybridization and differential immunologic screening of a T. pallidum genomic library. Both approaches led to the identification of a polymorphic multicopy gene family with predicted amino acid homology to the major sheath protein of Treponema denticola. One of the members of this gene family, tpr K, codes for a protein that is predicted to have a cleavable signal peptide and be located in the outer membrane of the bacterium. Reverse transcription polymerase chain reaction analysis of T. pallidum reveals that Tpr K is preferentially transcribed in the Nichols strain of T. pallidum. Antibodies directed to purified recombinant variable domain of Tpr K can opsonize T. pallidum, Nichols strain, for phagocytosis, supporting the hypothesis that this portion of the protein is exposed at the surface of the treponeme. Immunization of rabbits with the purified recombinant variable domain of Tpr K provides significant protection against infection with the Nichols strain of T. pallidum. This gene family is hypothesized to be central to pathogenesis and immunity during syphilis infection.

Amino Acid Sequence↗
PubMed1950

Studies on the life cycle of spirochetes; the life cycle of the Nichols pathogenic Treponema pallidum in the rabbit testis as seen by phase contrast microscopy.

A series of observations with the phase contrast microscope on the occurrence of a complex life cycle in the pathogenic Treponema pallidum as it occurs in the syphilitic rabbit testis has been presented and it seems likely from these observations that there are two means of vegetative reproduction, consisting of (1) transverse division (the most important under usual conditions); and (2) the production of gemmae or buds which eventuate into unispirochetal cysts comparable to those described for saprophytic forms, within each of which single spirochetes develop and differentiate, and from which they subsequently emerge. In addition preliminary evidence is presented which suggests that a more complex process is involved in which multispirochetal cysts develop following aggregation of two or more organisms. Within each of these larger cysts numerous organisms develop and subsequently emerge as tangled ropes. Following emergence, they subsequently undergo transverse division and gemmae formation, and so reproduce vegetatively. Subsequent papers will elaborate upon these processes.

Animals↗
PubMed1999

Identification of Treponema pallidum subspecies pallidum in a 200-year-old skeletal specimen.

Treponema pallidum subsp. pallidum, the causative agent of venereal syphilis, was detected in a 200-year-old skeletal specimen from Easter Island. An initial diagnosis of treponemal infection was confirmed by extensive purification of immunoglobulin that reacted strongly with T. pallidum antigen. Extracted DNA exhibited a single-base polymorphism that distinguished T.p. subsp. pallidum from 4 other human and nonhuman treponemes. Extensive precautions against contamination of the subject matter with modern treponemal DNA were employed, including analysis of archaeological and modern specimens in 2 geographically separate laboratories. Molecular determination of historical disease states by using skeletal material can significantly enhance our understanding of the pathology and spread of infectious diseases.

Antigens, Bacterial↗
PubMed2000

Virulent Treponema pallidum, lipoprotein, and synthetic lipopeptides induce CCR5 on human monocytes and enhance their susceptibility to infection by human immunodeficiency virus type 1.

Treponema pallidum, its membrane lipoproteins, and synthetic lipoprotein analogues (lipopeptides) were each examined to determine whether they induced CCR5 expression on human peripheral blood mononuclear cells (PBMC). Reverse transcription-polymerase chain reaction for CCR5 gene transcripts, macrophage inflammatory protein (MIP)-1beta binding assays, and flow cytometry revealed that either T. pallidum, a representative treponemal lipoprotein, or a corresponding synthetic lipopeptide induced CCR5 on CD14 monocytes but not on CD3 lymphocytes. CXCR4, the coreceptor for T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), was not induced on PBMC by treponemes or by lipoproteins or lipopeptides. Consistent with these findings, T. pallidum, lipoprotein, and synthetic lipopeptide all promoted the entry of a macrophage-tropic, but not a T cell-tropic, strain of HIV-1 into monocytes. These combined results imply that T. pallidum and its constituent lipoproteins likely induce the expression of CCR5 on macrophages in syphilitic lesions, thereby enhancing transmission of macrophage-tropic HIV-1.

Adult↗