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Treponema denticola cystalysin catalyzes beta-desulfination of L-cysteine sulfinic acid and beta-decarboxylation of L-aspartate and oxalacetate.

Pyridoxal 5'-phosphate-dependent cystalysin from Treponema denticola catalyzes the beta-displacement of the beta-substituent from both L-aspartate and L-cysteine sulfinic acid. The steady-state kinetic parameters for beta-desulfination of L-cysteine sulfinic acid, k(cat) and K(m), are 89+/-7 s(-1) and 49+/-9 mM, respectively, whereas those for beta-decarboxylation of L-aspartate are 0.8+/-0.1 s(-1) and 280+/-70 mM. Moreover, cystalysin in the pyridoxamine 5'-phosphate form has also been found to catalyze beta-decarboxylation of oxalacetate as shown by consumption of oxalacetate and a concomitant production of pyruvate. The k(cat) and K(m) of this reaction are 0.15+/-0.01 s(-1) and 13+/-2 mM, respectively. Possible mechanistic and physiological implications are discussed.

Animals↗

Diagnosis of congenital syphilis from placental examination: comparison of histopathology, Steiner stain, and polymerase chain reaction for Treponema pallidum DNA.

Congenital syphilis is often a presumptive diagnosis (based on serologies), because confirmation requires identification of Treponema pallidum in fetal/neonatal tissues or in the placenta. Placental histological features associated with congenital syphilis include the triad of enlarged hypercellular villi, proliferative fetal vascular changes, and acute or chronic villitis. The authors blindly evaluated 49 formalin-fixed, paraffin-embedded placentas (38 with positive maternal syphilis serologies; 11 with negative serologies) and compared results of histology, Steiner stain, and polymerase chain reaction (PCR) for T pallidum DNA. Histology was categorized as positive (triad present), suspicious (two thirds of triad present), or negative. Treponemal DNA was detected by amplifying a 189 base pair region of the 47 kd treponemal membrane antigen with 44 cycles of PCR; products were detected by Southern blot. Placentas from the 11 seronegative mothers were all negative by histology, Steiner stain, and PCR. Among the 38 placentas from serologically positive mothers, 4 had positive histology (2 of 4 positive Steiner, 4 of 4 positive PCR); 6 had suggestive histology (0 of 6 positive Steiner; 1 of 6 positive PCR); and, 28 had negative histology (0 of 28 positive Steiner; 1 of 28 positive PCR). PCR identification of treponemal DNA was significantly associated with the triad (P = .0003), proliferative fetal vascular changes (P = .0003), acute villitis (P = .003), chronic villitis (P = .004), and spirochetes on Steiner stain (P = .01). These results (1) confirm a strong association between placental histopathologic features and congenital syphilis; (2) indicate that when such features are present, PCR of placental tissue may confirm the diagnosis of congenital syphilis; and (3) suggest that even when such features are absent, PCR of placental tissue may identify additional cases of histologically unsuspected congenital syphilis.

Base Sequence↗

Demonstration of locally synthesized immunoglobulin M antibodies to Treponema pallidum in the central nervous system of patients with untreated neurosyphilis.

Immunoglobulin M (IgM) antibodies to Treponema pallidum (TP) in serum and cerebrospinal fluid (CSF) can be evaluated quantitatively using a recently introduced enzyme-linked immunosorbent assay (ELISA). In 19 patients with untreated neurosyphilis, local synthesis of TP-specific IgM antibodies within the central nervous system (CNS) was demonstrated by estimation of the CSF/serum ratio of the TP-IgM-ELISA titer/mg total IgM. The TP-specific IgM antibody levels/mg total IgM in the CSF were between 3- and 75-fold higher than those in the corresponding serum of the same patients. Patients with untreated syphilis but without CNS involvement showed equal amounts of TP-specific IgM antibody levels/mg total IgM in CSF and serum, and served as controls.

Adult↗

Recombinant Treponema pallidum antigens in syphilis serology.

Treponema pallidum, the etiological agent of syphilis, is characterized by a paucity of surface exposed outer membrane proteins and a high content of cytoplasma membrane associated lipoproteins. At all stages of infection intense antibody responses against lipoproteins are detectable. In order to provide antigens for syphilis diagnosis the highly immunogenic lipoproteins TpN17, TpN29-35 (TpD), TpN44.5 (TmpA), TpN47, and TpN35 (TmpC) and the membrane protein TpN39 (BMP) were cloned. Insertion of PCR amplified DNA into an E. coli expression vector resulted in high level expression of antigens. N-terminal hexahistidine sequence allowed efficient purification of fusion proteins by metal chelate affinity chromatography. The recombinant antigens were tested in enzyme-linked immunosorbent assays. TpN17, TpN47, and TpN44.5 antigens showed high antibody titers. Assays with the three antigens combined resulted in a further improvement of diagnostic sensitivity in comparison with single antigens. Antibodies were found in 17 of 18 patients in all stages of syphilis, whereas 42 normal human sera were nonreactive. No cross-reactivity was detected in 24 sera of patients with Lyme borreliosis.

Antigens, Bacterial↗

Eliminating organisms against Treponema hyodysenteriae in the gut of mice.

Ta:CF#1 mice lost their high susceptibility to Treponema hyodysenteriae infection after being mingled with Slc:ICR mice which had no susceptibility to the infection. Ta:CF#1 mice also lost susceptibility to the infection after being treated orally with fecal suspensions from Slc:ICR mice; treatment with heated (70 degrees C, 15 min) or filtered (pore size 0.2 micron) fecal suspensions did not cause a loss of susceptibility. Furthermore, even after the infection had established, Ta:CF#1 mice became free from T. hyodysenteriae and cecal lesions by oral treatment with the fecal suspensions. When Slc:ICR mice were treated with spectinomycin throughout the experiment, they became susceptible to the infection. These studies indicate that there exist eliminating organisms against T. hyodysenteriae in the gut of mice.

Animals↗

Investigations into the location of Treponema hyodysenteriae in the cecum of experimentally infected young broiler chicks by light- and electronmicroscopy.

Numerous Treponema hyodysenteriae were present both on the mucosal surface and in the deep crypts of the cecum of young broiler chicks 7 and 14 days after inoculation with the treponemes. The treponemes in the ceca of chicks inoculated with 10(8) cells were observed more frequently than those of chicks inoculated with 10(7) cells. The treponemes in the ceca were observed by light microscopy, scanning electron microscopy and transmission electron microscopy. The lesions were primarily confined to the cecum. Desquamation of epithelial cells, edema, leukocytic infiltration and hemorrhage were observed in the mucosae.

Animals↗

Unusual manifestations of secondary syphilis and abnormal humoral immune response to Treponema pallidum antigens in a homosexual man with asymptomatic human immunodeficiency virus infection.

The case presented here involves a 32-year-old homosexual man with human immunodeficiency virus (HIV) seropositivity and unusual manifestations of secondary syphilis. The patient presented with syphilitic keratoderma and chorioretinitis, and his appearance superficially resembled that of a patient with Reiter's syndrome. Although nontreponemal and treponemal tests for syphilis showed reactivity, the patient's humoral immune response to individual polypeptides of Treponema pallidum, measured by Western blot analysis, was markedly abnormal. The possible relationship between asymptomatic HIV infection and an abnormal humoral immune response to a second pathogen, in this case T. pallidum, is discussed. Our case is one of several recent cases of active syphilis reported in individuals with HIV seropositivity.

Adult↗

Syphilitic gastritis: demonstration of Treponema pallidum with the use of fluorescent treponemal antibody absorption complement and immunoperoxidase stains.

Because of the decrease in the overall incidence of syphilis, syphilitic involvement of stomach is seldom reported in the modern literature. Because of the nonspecific symptoms and signs of the disease, it is necessary to demonstrate Treponema pallidum in the gastric lesions to confirm the diagnosis. With the use of immunofluorescence and immunoperoxidase methods we have succeeded in identifying T. pallidum in the gastric wall of a patient who initially had cutaneous manifestations of secondary syphilis and gastric symptoms.

Adult↗

Purification and characterization of Treponema hyodysenteriae hemolysin.

A hemolysin produced by Treponema hyodysenteriae ATCC27164 was purified from broth filtrates by acetic and (NH4)2SO4 precipitations followed by ion exchange chromatography on diethylaminoethyl-Sephacel and gel filtration using Ultrogel AcA44. The purified hemolysin displayed only one band on polyacrylamide gel electrophoresis. By gel filtration the molecular weight was estimated as 74,000 daltons. The isolated hemolysin was oxygen resistant, heat labile and was not inactivated over a wide range of pH values. Further analysis indicated that this hemolysin was probably a polypeptide or a protein associated with lipids and nucleotides. Its action on rabbit erythrocytes which did not require any divalent cations could not be related to a lipolytic or proteolytic activity.

Animals↗

DNA probability profiles: examples from the Treponema pallidum genome.

In this paper we apply an algorithm developed by Poland (Biopolymers 13 (1974) 1859) to treat the statistical mechanics of the thermal unwinding of DNA to the genome of Treponema pallidum, the syphilis spirochete. We calculate probability profiles (giving the probability that each unit in the molecule is in the helix-state) and other statistical distributions for genes and sequences of genes, the longest containing 100 genes and 107,139 base pairs (approximately 10% of the genome).

Algorithms↗

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.

Adhesins, Bacterial↗

Cloning and expression of a pectate lyase from the oral spirochete Treponema pectinovorum ATCC 33768.

The pelA gene, encoding a pectate lyase, from Treponema pectinovorum ATCC 33768 was isolated by heterologous expression of a cosmid library in Escherichia coli. In vitro transposon mutagenesis identified an open reading frame of 1293 bp capable of encoding a protein of 430 amino acids with a predicted amino-terminal signal sequence of 21 amino acids. Analysis of the amino acid sequence suggested that it is a member of the polysaccharide lyase family 10 of which all characterized members show pectate lyase activity. An amino-terminal His-tagged recombinant form of PelA was expressed and purified from E. coli. The recombinant enzyme has characteristics common to other bacterial pectate lyases such as an alkaline pH optimum, dependence on calcium ions for activity, and inhibition by zinc ions.

Amino Acid Sequence↗

A 43-kDa protein of Treponema denticola is essential for dentilisin activity.

A protease of Treponema denticola, dentilisin, is thought to be part of a complex with 43- and 38-kDa proteins. A sequence encoding a 43-kDa protein was located in the 3' region of the prcA gene upstream of the dentilisin gene (prtP). The 43-kDa protein was apparently generated from digestion of PrcA. To clarify the function of the protein, we constructed a mutant of the 43-kDa protein following homologous recombination. The mutant lacked detectable dentilisin activity. Immunoblot analysis demonstrated that the dentilisin protein was degraded in the mutant. The results of real-time polymerase chain reaction suggested that prtP mRNA expression in the mutant was somewhat decreased compared with the wild-type strain. These data suggest that the 43-kDa protein is involved in the stabilization of the dentilisin protein.

Bacterial Proteins↗

Molecular characterization of the gyrB region of the oral spirochete, Treponema denticola.

The nucleotide (nt) sequence of the Treponema denticola (Td) DNA gyrase beta-subunit gene (gyrB) has been determined. Southern blot analysis of Td chromosomal DNA indicated that gyrB is present as a single copy. Approximately 3.2kb of the nt sequence 5' and 0.7kb of nucleotide sequence 3' of gyrB were obtained. Analysis of the deduced amino acid (aa) sequence revealed two complete open reading frames (ORFs) (ORF1 and ORF3) and a truncated ORF (ORF4'). ORF1 has no homology to sequences in the databases, whereas ORF3 and ORF4' have significant homology to several bacterial DnaA (replication initiator) and DnaE (DNA polymerase III) proteins respectively. RT-PCR data showed that orf1-gyrB are co-transcribed, while dnaA-dnaE are co-transcribed but in the opposite direction. These data indicated that the gene organization of the Td gyrB region is unique compared with that of other bacteria. Eighteen putative DnaA boxes with several AT-rich regions were identified in the dnaA-dnaE intergenic region, and three putative DnaA boxes were identified in the gyrB-dnaA intergenic region. Spontaneous coumermycin A(1)-resistant Td mutants were isolated and characterized. The mutants have a >20-fold higher resistance to coumermycin A(1) than wild-type Td. A single point mutation in gyrB that changed GyrB Lys(136) to Glu or Thr appears to be responsible for the coumermycin A(1) resistance.

Amino Acid Sequence↗

Identification and transcriptional analysis of a Treponema pallidum operon encoding a putative ABC transport system, an iron-activated repressor protein homolog, and a glycolytic pathway enzyme homolog.

We have characterized a 5.2-kilobase (kb) putative transport related operon (tro) locus of Treponema pallidum subsp. pallidum (Nichols strain) (Tp) encoding six proteins: TroA, TroB, TroC, TroD, TroR and Phosphoglycerate mutase (Pgm). Four of these gene products (TroA-TroD) are homologous to members of the ATP-Binding Cassette (ABC) superfamily of bacterial transport proteins. TroA (previously identified as Tromp1) has significant sequence similarity to a family of Gram-negative periplasmic substrate-binding proteins and to a family of streptococcal proteins that may have dual roles as substrate binding proteins and adhesins. TroB is homologous to the ATP-binding protein component, whereas TroC and TroD are related to the hydrophobic membrane protein components of ABC transport systems. TroR is similar to Gram-positive iron-activated repressor proteins (DesR, DtxR, IdeR, and SirR). The last open reading frame (ORF) of the tro operon encodes a protein that is highly homologous to the glycolytic pathway enzyme, Pgm. Primer extension results demonstrated that the tro operon is transcribed from a sigma 70-type promoter element. Northern analysis and reverse transcriptase-polymerase chain reactions provided evidence for the presence of a primary 1-kb troA transcript and a secondary, less abundant, troA-pgm transcript. The tro operon is flanked by a Holliday structure DNA helicase homolog (upstream) and two ORFs representing a purine nucleoside phosphorylase homolog and tpp15, a previously characterized gene encoding a membrane lipoprotein (downstream). The presence of a complex operon containing a putative ABC transport system and a DtxR homolog indicates a possible linkage between transport and gene regulation in Tp.

ATP-Binding Cassette Transporters↗

Amplification of the DNA polymerase I gene of Treponema pallidum from whole blood of persons with syphilis.

Previous reports suggest that Treponema pallidum bacteremia occurs in persons with syphilis exposure ('incubating syphilis') and in persons with primary or secondary syphilis. During a recent syphilis outbreak, whole blood samples from 32 persons with suspected syphilis or syphilis exposure were screened using polymerase chain reaction (PCR) to amplify the DNA polymerase I gene (polA) of T. pallidum. Of the 32 samples, polA was amplified from 13 (41%). Of these 13, three were determined to have incubating syphilis; two had primary or secondary syphilis and eight had latent syphilis. This study demonstrates that spirochetemia can occur throughout the course of T. pallidum infection.

Adult↗

Construction and analysis of hemin binding protein mutants in the oral pathogen Treponema denticola.

Treponema denticola, a periodontal pathogen, can use hemin as its sole iron source. The organism synthesizes two low-iron-induced outer-membrane hemin-binding proteins, HbpA and HbpB. To characterize genetically the function of these two novel proteins, standard recombinant DNA procedures and electroporation were used to construct T. denticola strains in which the genomic copies of either hbpA or both hbpA and hbpB were interrupted with an erythromycin resistance cassette. Northern blot and RT-PCR analyses verified that the normal hbpA transcripts were missing in both mutants. The hbpA mutation also had a polar effect on the transcription of hbpB and thus neither mutant strain transcribes the downstream hbpB gene. The parental and hbp mutant strains had similar growth properties in normal media, but the mutants reached a lower cell density than parental cells in iron-restricted media. The results indicate that HbpA and/or HbpB are required for efficient iron utilization but that there is an additional system that can help T. denticola acquire iron. The growth defect of the mutants was totally restored by lactoferrin but only partially restored by adding exogenous hemin or inorganic iron. Thus, hbpA and/or hbpB specifically facilitate hemin and iron utilization under low iron conditions and are presumably important for T. denticola virulence in the host environment.

Bacterial Outer Membrane Proteins↗

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.

Animals↗