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.     

Multiple alleles of Treponema pallidum repeat gene D in Treponema pallidum isolates

Two new tprD alleles have been identified in Treponema pallidum: tprD2 is found in 7 of 12 T. pallidum subsp. pallidum isolates and 7 of 8 non-pallidum isolates, and tprD3 is found in one T. pallidum subsp. pertenue isolate. Antibodies against TprD2 are found in persons with syphilis, demonstrating that tprD2 is expressed during infection.     

Treponema pallidum fibronectin-binding proteins

Putative adhesins were predicted by computer analysis of the Treponema pallidum genome. Two treponemal proteins, Tp0155 and Tp0483, demonstrated specific attachment to fibronectin, blocked bacterial adherence to fibronectin-coated slides, and supported attachment of fibronectin-producing mammalian cells. These results suggest Tp0155 and Tp0483 are fibronectin-binding proteins mediating T. pallidum-host interactions.     

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.     

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.     

Shape of Treponema pallidum

Treponema pallidum was found to be not helical, but a flat wave twisted into one to five different planes per cell.     

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.     

Organization of the ribosomal RNA genes in Treponema phagedenis and Treponema pallidum.

The genomic DNA fragment which contains ribosomal RNA (rRNA) genes for Treponema phagedenis was cloned into bacteriophage vector lambda EMBL3. A restriction map of the fragment was constructed and the organization of the rRNA genes was determined. The fragment contained at least one copy of the 16S, 23S and 5S sequences and the genes are arranged in the order 16S-23S-5S. Southern hybridization using radiolabeled rRNA gene probes to genomic DNA from T. phagedenis strain Reiter and T. pallidum strain Nichols showed that these organisms have two radioactive fragments which hybridize to the probes in their genome. These results suggest that both pathogenic and non-pathogenic strains of Treponema may carry at least two sets of rRNA genes on their chromosomes.     

Genetics of Treponema: relationship between Treponema pallidum and five cultivable treponemes.

Three genetically distinct groups of treponemes have been identified by saturation reassociation assays using 125I-labeled treponemal DNAs. The three groups are (i) virulent Treponema pallidum (Nichols strain), (ii) T. phagedenis and its biotypes Reiter and Kazan 5, and (iii) T. refringens biotypes Nichols and Noguchi. There is no detectable DNA sequence homology (less than 5%) among the three groups. The groups have distinct guanine + cytosine contents: 52.4 to 53.7% for T. pallidum, 41.5% for T. refringens, and 38 to 39% for T. phagedenis.     

Genetic relationship between Treponema pallidum and Treponema pertenue, two noncultivable human pathogens.

Genetic relationships among two strains of Treponema pallidum (Nichols and KKJ) and a strain of T. pertenue were determined by measuring the degree of deoxyribonucleic acid sequence homology. The results in indicated that these three virulent, noncultivable treponemes were genetically indistinguishable. Like T. pallidum (Nichols), T. pertenue (Gauthier) had no detectable deoxyribonucleic acid sequence homology with T. phagedenis (biotype Reiter), T. refringens (biotype Noguchi), or with salmon sperm.     

Locomotory characteristics of Treponema denticola

Locomotion of pathogenic spirochetes has been suggested as a virulence factor in their pathogenesis. Little is known of the locomotory characteristics of oral anaerobic spirochetes. We have determined the optimal conditions for motility of seven strains of Treponema denticola in menstrua of different viscosities. The viscosity for optimum motility for all strains was found to be 9.57 centipoises at 25 degrees C. Under these conditions the average speeds for each strain was computed from the motility tracks as recorded by timed exposures under dark-field microscopy. Differences in speeds were found between the various strains. In addition, we have determined the "persistence" (direct distance/actual pathlength travelled) of cell movement of each strain. Interstrain differences were also noted. These locomotory characteristics contribute to the locomotory phenotypes of the various strains and therefore may aid in their characterization and provide an insight into locomotion as a virulence factor in periodontitis.     

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.