Fatty acid requirement of Treponema denticola and Treponema vincentii

Treponema denticola and Treponema vincentii were cultured in a medium supplemented with either 0.2 or 0.4% (w/v) alpha globulin in place of serum. The active factor(s) in alpha globulin was stable at pH 7.0 to autoclaving and was nondialyzable. Extraction of lipids from alpha globulin showed that both protein and lipid, supplied by the alpha globulin, were required for maximal growth of these two oral treponemes. The lipid component was investigated by adding sodium salts of long-chain fatty acids to the basal medium supplemented with 0.4% delipified alpha globulin. The lipid component of alpha globulin was replaced by either oleic acid (cis-18:1(9)) or by elaidic acid (trans- 18:1 (9)0. No other saturated or unsaturated fatty acid tested could support good growth. Tween 80 (polysorbitan monooleate) was the only Tween compound able to support maximal growth of T. denticola. The cellular lipids of T. denticola, grown with oleate in broth supplemented with 0.4% delipified alpha globulin, were extracted and analyzed by gas chromatography. The principle fatty acids were myristic, pentadecanoic, and palmitic acids. Lesser amounts of oleic acid, eicosadienoic acid, and an unidentified fatty acid (retention time, 88 min) were also detected. Treponema denticola appears to be capable of limited synthesis of cellular fatty acids such as myristic, pentadecanoic, and palmitic acids from oleic acid.     

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.     

Antigenic and structural analysis of Treponema denticola

Polypeptide and Western immunoblot profiles of subcellular fractions of Treponema denticola ATCC 33520 have been determined by SDS-PAGE of Triton X-100-soluble and -insoluble fractions, a lipopolysaccharide-enriched fraction and purified flagella. Major Triton X-100-soluble polypeptides of 72, 68, 54 and 52 kDa were detected. The 54 kDa polypeptide appeared to be a breakdown product of a larger, heat-modifiable polypeptide. Based on the results of SDS-PAGE analysis and immunoblotting of proteinase K digests of T. denticola, a 'rough' lipopolysaccharide appeared to be present. Electron microscopy has been used to monitor the effect of detergent treatment on the morphology of the organism and to examine the detailed structure of the flagella. Treatment with Triton removed the T. denticola outer membrane, resulting in exposure of the flagella. The flagella were shown to have a complex sheath and core structure and polypeptide composition characteristic of that observed for other treponemes. Polypeptides of 38, 35, 32 and 28 kDa were present in purified flagella preparations. Immunoelectron microscopy, iodine-labelling and Western blotting were used to demonstrate the exposure of antigens on the T. denticola surface. Surface iodination located polypeptides of 72, 68 and 54 kDa. Antiserum raised against whole cells of T. denticola recognized these polypeptides and an additional polypeptide of 52 kDa. These data provide a basis for future detailed molecular analysis of the ultrastructure and antigenicity of T. denticola.     

Development of quasi-multicellular bodies of Treponema denticola

The formation of quasi-multicellular bodies of Treponema denticola was analysed using different electron microscopical methods. These bacteria could develop four different conformations: (i) normal helical forms; (ii) twisted spirochetes, forming plaits; (iii) twisted spirochetes, forming club-like structures; (iv) spherical bodies in different size. Treponemes within spherical bodies, plaits, and clubs proved to be enclosed in a common outer sheath in which the normal arrangement of their axial flagella was lost. The development of the quasi-multicellular bodies starting from the monoforme spirochetes was elucidated and this morphogenetic process is illustrated by a schematic drawing. Factors which might be involved in the induction of the structures are discussed and their possible pathogenetic importance is considered.     

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lacto-ferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.     

Cleavage of Human Immunoglobulin G by Treponema denticola

Mechanisms by which microbial proteases may counteract the local host immune system include the degradation of immunoglobulins. In this study, we report the capacity of the periodontopathogen Treponema denticola to degrade immunoglobulin G (IgG). Intact IgG was not hydrolysed by whole cells, as revealed by SDS-PAGE analysis. When IgG molecules were treated with endoglycosidase F to remove the carbohydrate moiety, significant degradation was observed. However, pre-treatment with glycosidases possessing specificities different from endoglycosidase F (lysozyme or neuraminidase) did not render the molecule susceptible to cleavage by T. denticola. SDS-PAGE analysis of the IgG degradation products suggests that T. denticola cleaves inside the heavy chain polypeptide. Serine-specific protease inhibitors were highly effective in inhibiting the degradation of glycosidase-treated IgG molecules by T. denticola. The synergistic effect of glycolytic enzymes andT. denticola proteases on IgG may occur during periodontitis since both glycolytic activities and spirochete numbers significantly increase in diseased periodontal sites.     

Growth stimulation of Treponema denticola by periodontal microorganisms

Previous experiments have indicated that enrichment of subgingival plaque in human serum can lead to the accumulation of Treponema denticola. T. denticola depends on bacterial interactions for its growth in serum. Aim of the present study was to identify specific microorganisms involved in the growth stimulation of T. denticola. To this end, strains isolated from previous plaque enrichment cultures were tested for growth stimulation in co-cultures with T. denticola. In addition, growth of T. denticola was tested in culture filtrates of the same strains, Bacteroides intermedius, Eubacterium nodatum, Veillonella parvula and Fusobacterium nucleatum were found to enhance growth of T. denticola in co-cultures. A continuous co-culture of T. denticola, F. nucleatum and B. intermedius in human serum gave very high levels of T. denticola, up to 3.10(9).ml-1. Mechanisms involved in growth stimulation may include the ability of B. intermedius and E. nodatum to cleave the protein-core of serum (glyco-)proteins, making these molecules accessible for degradation by T. denticola. In addition, E. nodatum was found to produce a low-molecular weight growth-factor for T. denticola, that was heat-stable and acid as well as alkaline resistant. V. parvula may provide peptidase activities complementary to those of T. denticola. The nature of the growth enhancing activity of F. nucleatum is yet unknown. The data support the dependency of T. denticola on other bacterial species for growth in the periodontal pocket.     

Treponema denticola PurE Is a bacterial AIR carboxylase

De novo purine biosynthesis proceeds by two divergent paths. In bacteria, yeasts, and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxy-AIR (CAIR) by two enzymes: N(5)-carboxy-AIR (N(5)-CAIR) synthetase (PurK) and N(5)-CAIR mutase (class I PurE). In animals, the conversion of AIR to CAIR requires a single enzyme, AIR carboxylase (class II PurE). The CAIR carboxylate derives from bicarbonate or CO(2), respectively. Class I PurE is a promising antimicrobial target. Class I and class II PurEs are mechanistically related but bind different substrates. The spirochete dental pathogen Treponema denticola lacks a purK gene and contains a class II purE gene, the hallmarks of CO(2)-dependent CAIR synthesis. We demonstrate that T. denticola PurE (TdPurE) is AIR carboxylase, the first example of a prokaryotic class II PurE. Steady-state and pre-steady-state experiments show that TdPurE binds AIR and CO(2) but not N(5)-CAIR. Crystal structures of TdPurE alone and in complex with AIR show a conformational change in the key active site His40 residue that is not observed for class I PurEs. A contact between the AIR phosphate and a differentially conserved residue (TdPurE Lys41) enforces different AIR conformations in each PurE class. As a consequence, the TdPurE·AIR complex contains a portal that appears to allow the CO(2) substrate to enter the active site. In the human pathogen T. denticola, purine biosynthesis should depend on available CO(2) levels. Because spirochetes lack carbonic anhydrase, the corresponding reduction in bicarbonate demand may confer a selective advantage.     

Treponema denticola infection is not a cause of false positive Treponema pallidum serology

It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but so far there are no definitive data supporting this hypothesis. In this study we tested 250 serum samples obtained from blood donors. All of them were negative when routinely screened for antibodies against Treponema pallidum. Then, all these samples were tested by immunoenzymatic (ELISA) and Western Blot (WB) assays to investigate reactivities against T. denticola. Thirteen samples showed a strong positivity when tested by both methods. When tested by WB against T. pallidum no sample met the positivity criteria. Nevertheless, bands with molecolar weights of about 30-35 KDa (endoflagellar core antigens) were recognized. All the 13 subjects serologically T. denticola positive underwent oral clinical and radiological observation: all showed a very poor parodontal status (CPSS > 103). Eleven crevicular fluid samples out of the total of 13 patients were T. denticola positive by Real Time PCR carried out using a LightCycler system. In this study we demonstrated that the presence of T. denticola in the crevicular fluid samples obtained from patients with a severe periodontal status and/or a positive serology against T. denticola is not a cause of false positive results in syphilis serology.     

Physical map of the Treponema denticola circular chromosome.

A physical map of the Treponema denticola ATCC 33520 genome was constructed by pulsed-field gel electrophoresis and DNA hybridization. The organism possesses a single, circular chromosome of approximately 3.0 Mbp and a 2.6-kbp circular plasmid, pTD1. The physical map of the A+T-rich genome was constructed with the rare-cutting restriction enzymes AscI, NotI, and SrfI, which have 8-bp G+C-rich recognition sites. The genes flgE, tdpA, and prtB encoding the flagellar hook protein, a 53-kDa immunogenic protein, and chymotrypsinlike protease, respectively, were located on the map. This treponeme was found to have two copies of each of the rRNA genes, as has been found to be the case for both Treponema phagedenis and Treponema pallidum.     

Amino acid and glucose fermentation by Treponema denticola

Summary Treponema denticola was grown in serum-containing media to which ¹⁴C-labelled compounds were added. Determinations of radioactivity in the products formed indicated that the organism fermented alanine, cysteine, glycine, serine, and glucose. Fermentation products included acetate, lactate, succinate, formate, pyruvate, ethanol, CO₂, H₂S, and NH₃. The products formed from glucose constituted a small portion of the total products. Assays of enzymatic activities in cell extracts indicated that the organism degraded glucose via the Embden-Meyerhof pathway. T. denticola possessed a coenzyme A-dependent CO₂-pyruvate exchange activity associated with a clostridial-type clastic system for pyruvate metabolism. Phosphotransacetylase and acetate kinase activities were present in cell extracts. Acetyl phosphate formation and benzyl viologen reduction were detected when cell extracts were incubated with pyruvate, serine or cysteine. The data indicate that T. denticola is an amino acid fermenter and that it possesses the enzymes needed for the fermentation of glucose. However, glucose does not serve as the primary substrate when the organism grows in media including both this carbohydrate and amino acids.     

Isolation and characterization of a plasmid from Treponema denticola

Agarose gel electrophoresis of whole genomic DNA of the oral spirochaete Treponema denticola has revealed a plasmid-like fraction. Purification and restriction enzyme analysis has confirmed the presence of a 2.6-kb circular plasmid, which has been mapped for restriction sites and cloned into the Escherichia coli plasmid pUC18. Southern blot analysis of genomic T. denticola DNA, using the plasmid as a probe, has shown that the plasmid is present only as an extra-chromosomal element. No plasmid-coded recombinant gene product from a PstI insert in pUC18 has been detected in host cells of E. coli by SDS-PAGE or immunoblotting with polyclonal immune rabbit serum to T. denticola. The discovery of this plasmid may provide a useful tool in the application of new molecular approaches in spirochaetal biology.     

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.     

Cytoplasmic filament-deficient mutant of Treponema denticola has pleiotropic defects

In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF and ermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpA insertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.     

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

Synergistic biofilm formation by Treponema denticola and Porphyromonas gingivalis

Biofilm formation is an important step in the etiology of periodontal diseases. In this study, in vitro biofilm formation by Treponema denticola and Porphyromonas gingivalis 381 displayed synergistic effects. Confocal microscopy demonstrated that P. gingivalis attaches to the substratum first as a primary colonizer followed by coaggregation with T. denticola to form a mixed biofilm. The T. denticola flagella mutant as well as the cytoplasmic filament mutant were shown to be essential for biofilm formation as well as coaggregation with P. gingivalis. The major fimbriae and Arg-gingipain B of P. gingivalis also play important roles in biofilm formation with T. denticola.     

Mitogenic activity of the outer membrane of Treponema denticola.

The outer membrane (OM) was isolated by detergent extraction from Treponema denticola ATCC 35405, ATCC 33521 and ATCC 35404, representing serovars a, b and c, respectively, as well as from two fresh isolates of T. denticola. Strict precautions were undertaken against the introduction of contaminant lipopolysaccharide when the OM was isolated. The OM was active in mitogenic stimulation of C3H/HeOuJ mouse spleen cultures, but to a somewhat lesser extent than purified lipopolysaccharide (LPS) from Escherichia coli 055:B5. Polymyxin B only partially inhibited the response. Unheated OM abrogated mitogenic activity of E. coli LPS, but heated preparations enhanced the mitogenic activity of E. coli LPS, suggesting the presence of a heat-labile cytolytic factor associated with T. denticola OM in addition to a putative lipopolysaccharide and/or heat-stable lipoprotein.     

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

The genomes of Treponema denticola and Treponema pallidum contain a gene, licCA, which is predicted to encode a fusion protein containing choline kinase and CTP:phosphocholine cytidylyltransferase activities. Because both organisms have been reported to contain phosphatidylcholine, this raises the possibility that they use a CDP-choline pathway for the biosynthesis of phosphatidylcholine. This report shows that phosphatidylcholine is a major phospholipid in T. denticola, accounting for 35-40% of total phospholipid. This organism readily incorporated [14C]choline into phosphatidylcholine, indicating the presence of a choline-dependent biosynthetic pathway. The licCA gene was cloned, and recombinant LicCA had choline kinase and CTP:phosphocholine cytidylyltransferase activity. The licCA gene was disrupted in T. denticola by erythromycin cassette mutagenesis, resulting in a viable mutant. This disruption completely blocked incorporation of either [14C]choline or 32Pi into phosphatidylcholine. The rate of production of another phospholipid in T. denticola, phosphatidylethanolamine, was elevated considerably in the licCA mutant, suggesting that the elevated level of this lipid compensated for the loss of phosphatidylcholine in the membranes. Thus it appears that T. denticola does contain a licCA-dependent CDP-choline pathway for phosphatidylcholine biosynthesis.