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PubMed1971

Hydrogenation of unsaturated fatty acids by Treponema (Borrelia) strain B 2 5, a rumen spirochete.

The time course of hydrogenation of linoleic acid to trans-11-octadecenoic acid was observed in a growing culture of Treponema (Borrelia) strain B(2)5. A conjugated fatty acid, cis-9, trans-11-octadecadienoic acid, was identified as an intermediate in the process. The isomerase responsible for the conversion of linoleic acid to the conjugated fatty acid was found to be associated with a particulate fraction characterized by a high protein and lipid content in a 2:1 ratio. Optimum pH for isomerase activity was found to be 7.0 in 0.05 m potassium phosphate buffer. No cofactor requirements could be demonstrated for the isomerase. The sulfhydryl inhibiting agents, iodoacetamide, N-ethylmaleimide, and p-chloromercuribenzoate, inhibited isomerase activity. Isomerase activity was also inhibited by the metal chelators, o-phenanthroline, alpha, alpha'-bipyridyl, ethylenediaminetetraacetic acid, and 8-hydroxyquinoline. Linoleic (Delta9, 12), linolenic (Delta9, 12, 15), and gamma-linolenic (Delta6, 9, 12) acids served as effective substrates for the isomerase; however, the derivatives of linoleic and linolenic acid did not.

Animals↗
PubMed1973

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.

Cell Wall↗
PubMed1974

Purification and characterization of axial filaments from Treponema phagedenis biotype reiterii (the Reiter treponeme).

Axial filaments have been purified from Treponema phagedenis biotype reiterii (the Reiter treponeme) and partially characterized chemically. The preparations consist largely of protein but also contain small amounts of hexose (3%). Filaments dissociate to subunits in acid, alkali, urea, guanidine, and various detergents. Amino acid analyses show an overall resemblance to other spirochetal axial filaments and to bacterial flagella. Dissociated filaments migrate as a single band upon acrylamide gel electrophoresis at pH 4.3 (in 4 M urea and 10 (3) M ethylenediaminetetraacetate) and at pH 12, but in sodium dodecyl sulfate gels, three bands are obtained under a wide variety of conditions. Two of these bands migrate very close together, with molecular weights of 33,000 +/- 500. The other band has a molecular weight of 36,500 +/- 500. Analysis of axial filaments by the dansyl chloride method yields both methionine and glutamic acid as amino terminal end groups. Sedimentation equilibrium measurements on dissociated axial filaments in 7 M guanidine hydrochloride yield plots of log C against varkappa(2) which vary with the speed and initial protein concentration used. Molecular weight values calculated from these plots are consistent with a model in which axial filament subunits are heterogeneous with respect to molecular weight in the approximate range of 32,000 to 36,000.

Amino Acid Sequence↗
PubMed1976

Arginine catabolism by Treponema denticola.

Treponema denticola, an anaerobe commonly present in the human mouth, ferments various amino acids and glucose. Amino acid analyses indicated that substrate amounts of arginine were utilized by T. denticola growing in a complex, serum-containing medium. Cell suspensions metabolized L-arginine to citrulline, NH3, CO2, proline, and small amounts of ornithine. CO2, NH3, ornithine, and proline were produced from L-citrulline by cell suspensions. Determinations of radioactivity in products formed from L-[U-14C]ornithine indicated that cell suspensions converted this amino acid to proline. Furthermore, proline was excreted by cells growing in a complex, arginine-containing medium. Arginine iminohydrolase (deiminase) and ornithine carbamoyltransferase activities were detected in T. denticola cell extracts. Carbamoylphosphate dissimilation by extracts yielded adenosine triphosphate. The data indicate that T. denticola derives energy by dissimilating L-argine via the arginine iminohydrolase pathway. However, unlike some of the other bacteria that utilize this pathway, T. denticola converts to proline much of the ornithine derived from L-arginine.

Arginine↗
PubMed1977

Scanning electron microscopy of Treponema pallidum (Nichols strain) attached to cultured mammalian cells.

This paper describes the attachment of Treponema pallidum (Nichols strain) to cultured mammalian cells as a visualized by scanning electron microscopy. Treponemes were incubated for 3 hr with cultured cells derived from normal rabbit testes or human skin epithelium, then fixed, processed with critical-point drying, and examined with a Cambridge Mark 2A scanning electron microscope. Large numbers of treponemes became attached to the cultured cells without altering the morphological integrity of the cultured cells. Attachment appeared to involve a very close physical proximity of treponemes to the cultured cells; at the site of attachment, no changes such as swelling or indentation of the cultured cell surface were observed. The addition of ruthenium red to the fixatives produced a treponemal-associated surface precipitate. This material, which is probably mucopolysaccharide and/or phospholipid, may be important in protecting the organisms against host defense mechanisms; in addition, it may be involved in the serological unresponsiveness of freshly prepared suspensions of T. pallidum.

Animals↗
PubMed1985

Genetic characterization and partial sequence determination of a Treponema pallidum operon expressing two immunogenic membrane proteins in Escherichia coli.

A detailed physical and genetic map of a previously cloned 5.5-kilobase segment of Treponema pallidum DNA is described. This segment expressed two proteins that are cell membrane associated in Escherichia coli. The structural genes of these treponemal membrane proteins, tmpA and tmpB, are coordinately expressed, and transcription in E. coli can start from at least two different treponemal promoters. The tmpA and tmpB proteins are the products of in vivo proteolytic cleavage from precursor proteins which are 2 and 4 kilodaltons larger, respectively, than the mature proteins. Because the sizes of the corresponding proteins produced in T. pallidum were identical to those of the mature membrane proteins in E. coli, we concluded that a similar proteolytic processing takes place in both E. coli and T. pallidum. Although tmpA and tmpB were controlled by the same transcription signals, tmpB was expressed to a higher extent than tmpA, and only the tmpB product could be overproduced by placing the left lambda promoter in front of the structural genes. The nucleotide sequence of the T. pallidum tmpA gene was established. This is the first T. pallidum gene sequenced. Codon usage and the nature of transcriptional and translational signals are discussed. The deduced amino acid sequence indicated the presence of a sequence that was characteristic for a signal peptide. This sequence information allowed the construction of hybrid genes coding for proteins having beta-galactosidase enzyme activity as well as TmpA epitopes. The enzyme-linked antigen was expressed at a high level in E. coli when transcriptional and translational signals from coliphage lambda were used. In this case the protein produced was a sandwich protein consisting of 21 amino acids of the lambda cro protein, 204 amino acids of the T. pallidum TmpA protein, and 1,020 amino acids of the E. coli lambda-galactosidase. The potential use of this enzyme-linked antigen for the serodiagnosis of syphilis is discussed.

Amino Acid Sequence↗
PubMed1986

Properties of an ordered ring structure formed by recombinant Treponema pallidum surface antigen 4D.

Ultrastructural and biochemical studies of a recombinant Treponema pallidum surface antigen designated 4D have been conducted due to its likely biological significance. Electron microscopy demonstrated that the 190-kilodalton (kDa) 4D molecule is an ordered ring structure of 10-nm diameter. The 90-kDa proteinase K-treated 4D is an ordered ring structure of 6-nm diameter. Evidence is presented that the 190-kDa ordered ring is maintained by noncovalent bonds; 19-kDa monomers can reassociate in vitro to reform a 190-kDa molecule. Amino acid composition analysis of 190-kDa 4D showed that the molecule is composed of 45% hydrophobic residues. Evidence relating the structure of the 4D ordered ring to its potential role in the pathogenesis of syphilis is discussed.

Amino Acids↗
PubMed1986

Treponema phagedenis has at least two proteins residing together on its periplasmic flagella.

Treponema phagedenis is an anaerobic, motile spirochete with several periplasmic flagella (PFs) at each cell end. This study provides the first genetic evidence that multiple protein species are associated with the PFs. In addition, these proteins were found to reside together on a given PF. Nonmotile mutants which lacked the PFs were isolated, and spontaneous revertants to motility regained the PFs. These results suggest that the PFs are involved in the motility of T. phagedenis. Isolated PFs had two major protein bands with molecular weights of 33,000 and 39,800, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots with monoclonal and polyclonal antibodies indicated that both proteins were absent in the PF mutants but present in the revertants. Immunoelectron microscopy revealed that the 39,800-molecular-weight protein was distributed along the entire PF. Immunoprecipitation analysis suggested that the 39,800- and 33,000-molecular-weight proteins were closely associated in situ.

Bacterial Proteins↗
PubMed1987

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.

Bacterial Proteins↗
PubMed1987

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.

Antigens, Bacterial↗
PubMed1988

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.

Bacterial Outer Membrane Proteins↗
PubMed1990

Isolation and characterization of a Treponema pallidum major 60-kilodalton protein resembling the groEL protein of Escherichia coli.

A native structure containing the major 60-kilodalton common antigen polypeptide (designated TpN60) was isolated from Treponema pallidum subsp. pallidum (Nichols strain) through a combination of differential centrifugation and sucrose density gradient sedimentation. Gel filtration chromatography indicated that this structure is a high-molecular-weight homo-oligomer of TpN60. Antisera to TpN60 reacted with the groEL polypeptide of Escherichia coli, as determined by immunoperoxidase staining of two-dimensional electroblots. Electron microscopy of the isolated complex revealed a ringlike structure with a diameter of approximately 16 nm which was very similar in appearance to the groEL protein. Comparison of the N-terminal amino acid sequence of TpN60 with the deduced sequences of the E. coli groEL protein, related chaperonin proteins from mycobacteria and Coxiella burnetti, the hsp60 protein of Saccharomyces cerevisiae, the wheat ribulose bisphosphate carboxylase-oxygenase-subunit-binding protein (alpha subunit), and the human P1 mitochondrial protein indicated sequence identity at 8 of 22 to 10 of 22 residues (36 to 45% identity). We conclude that the oligomer of TpN60 is homologous to the groEL protein and related chaperonins found in a wide variety of procaryotes and eucaryotes and thus may represent a heat shock protein involved in protein folding and assembly.

Amino Acid Sequence↗
PubMed1990

Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.

Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.

Blotting, Southern↗
PubMed1991

The bent-end morphology of Treponema phagedenis is associated with short, left-handed, periplasmic flagella.

Treponema phagedenis Kazan 5 is a spirochete with multiple periplasmic flagella attached near each end of the cell cylinder. Dark-field microscopy revealed that most of the cell is right-handed (helix diameter, 0.23 micron; helix pitch, 1.74 microns), and the ends appear bent. These ends could move and gyrate while the central part of the cell remained stationary. The present study examines the basis for the bent-end characteristic. Motility mutants deficient in periplasmic flagella were found to lack the bent ends, and spontaneous revertants to motility regained the periplasmic flagella and bent-end characteristic. The length of the bent ends (2.40 microns) was found to be similar to the length of the periplasmic flagella as determined by electron microscopy (2.50 microns). The helix diameter of the bent ends was 0.57 micron, and the helix pitch of the bent ends was 1.85 microns. The periplasmic flagella were short relative to the length of the cells (15 microns) and, in contrast to the reports of others, did not overlap in the center of the cell. Similar results were found with T. phagedenis Reiter. The results taken together indicate that there is a causal relationship between the bent-end morphology and the presence of short periplasmic flagella. We report the first three-dimensional description of spirochete periplasmic flagella. Dark-field microscopy of purified periplasmic flagella revealed that these organelles were left-handed (helix diameter, 0.36 microns; helix pitch, 1.26 microns) and only 1 to 2 wavelengths long. Because of a right-handed cell cylinder and left-handed periplasmic flagella along with bent ends having helix diameters greater than those of either the cell cylinder or periplasmic flagella, we conclude that there is a complex interaction of the periplasmic flagella and the cell cylinder to form the bent ends. The results are discussed with respect to a possible mechanism of T. phagedenis motility.

Cell Movement↗
PubMed1991

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.

Amino Acid Sequence↗
PubMed1992

Molecular genetic analysis of a class B periplasmic-flagellum gene of Treponema phagedenis.

Treponema phagedenis is a host-associated spirochete with multiple polypeptides making up its periplasmic flagella (PFs). Each PF has a 39-kDa polypeptide making up the sheath (class A PF polypeptide) and two to four antigenically similar 33- to 34-kDa polypeptide species making up the core (class B PF polypeptides). A genetic analysis of the PF-deficient mutants T-40 and T-55 has shown that the PFs are involved in motility. To better understand the synthesis and assembly of these complex organelles and to compare the PF genes with those of other spirochetes, we cloned and characterized the T. phagedenis flaB2 gene, which encodes one class B polypeptide. The flaB2 gene consists of an open reading frame of 858 nucleotides capable of encoding a protein of 31.5 kDa. The predicted amino acid sequence of the FlaB2 polypeptide was 92% identical to that of T. pallidum FlaB2, with a 76% identity at the nucleotide level. These results confirm previous immunological and N-terminal-sequence analyses which suggested that the PF genes are well conserved in the spirochetes. Primer extension analysis of T. phagedenis flaB2 indicated that the start site of transcription was 127 nucleotides upstream from the ATG initiation codon. Preceding the start site is a DNA sequence similar to the sigma 28 consensus promoter sequence commonly found associated with motility genes. Northern (RNA) blots probed with a segment of flaB2 DNA revealed a 1,000-nucleotide monocistronic transcript in the wild type and in PF-deficient mutants T-40 and T-55. DNA sequencing of most of the flaB2 gene of the mutants revealed no differences from the wild-type gene. Because the mutants fail to synthesize detectable class B PF polypeptides yet synthesize extensive amounts of flaB2 mRNA, PF synthesis in T. phagedenis is likely to involve regulation at the translational level.

Amino Acid Sequence↗
PubMed1992

Homology of a plasmid from the spirochete Treponema denticola with the single-stranded DNA plasmids.

The 2,647-bp nucleotide sequence of cryptic plasmid pTD1, isolated from the oral spirochete Treponema denticola, was determined. The sequence revealed two open reading frames, A and B, which encode polypeptides of 335 and 235 amino acids, respectively. Open reading frame A shows sequence similarity to genes that encode replication proteins from a group of plasmids common in gram-positive bacteria, which replicate via a single-stranded intermediate.

Amino Acid Sequence↗
PubMed1994

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.

Animals↗