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PubMed1996

Characterization of the Treponema denticola prtP gene encoding a prolyl-phenylalanine-specific protease (dentilisin).

A chymotrypsin-like protease from Treponema denticola ATCC 35405 was purified by chromatographic techniques. The purified enzyme consisted of three polypeptides (38, 43, and 72 kDa). The protease exhibited specificity for peptide bonds containing phenylalanine and proline at the P1 and P2 positions, respectively, and was classified as a serine protease on the basis of inhibition studies. Naturally occurring protease inhibitors such as alpha1-antitrypsin and alpha1-antichymotrypsin had no effect on enzymatic activity. The enzyme degraded fibronectin, alpha1-antitrypsin, and gelatin while weakly degrading the immunoglobulin G heavy chain and type IV collagen. N-terminal amino acid sequences were determined for the 43- and 72-kDa proteins. On the basis of these sequences, the genes coding for the 43- and 72-kDa proteins were isolated and sequenced. The open reading frame which codes for the 72-kDa protein was designated prtP. This gene consists of 2,169 bp and codes for a protein with an Mr of 77,471. The protein appeared to be composed of a signal peptide region followed by a prosequence and the mature protein domain. The deduced amino acid sequence exhibited similarity with that of the Bacillus subtilis serine protease subtilisin. The deduced properties of the sequence suggest that the 72-kDa protein is a chymotrypsin-like protease. However, the nature and function of the 43-kDa protein have not yet been determined.

Amino Acid Sequence↗
PubMed1996

Proline iminopeptidase from the outer cell envelope of the human oral spirochete Treponema denticola ATCC 35405.

Certain periodontopathic organisms have been shown to exhibit high activity of proline iminopeptidase (PIPase). The human oral spirochete Treponema denticola ATCC 35405 was found to contain an easily extractable, novel PIPase (EC 3.4.11.5), which was purified to a sodium dodecyl sulfate- polyacrylamide gel electrophoresis-pure form by means of fast protein liquid chromatographic procedures. The range of the minimum monomeric molecular mass (280 amino acid residues) of the PIPase, based on amino acid analysis, was 30.35 to 30.39 kDa, but the likely in vivo form of the enzyme is a tetramer (minimum mass, 120.2 to 120.4 kDa). The molecular masses based on laser desorption mass spectrometry were 36.058 kDa for the monomer and 72.596 kDa for a dimer. The PIPase cleaves specifically the Pro-Y bond in dipeptides where Y is preferably Arg or Lys. Pro-Gln, Pro-Asn, and Pro-Ala were also good substrates, while Pro-Glu was hydrolyzed slowly and Pro-Asp was not hydrolyzed at all. Tripeptides were poor substrates or were not hydrolyzed (an exception was Pro-Gly-Gly, which cleaved at a moderate rate). Larger molecules, such as poly-L-Pro, were not hydrolyzed. The T. denticola enzyme can be regarded as a true PIPase, since replacing Pro in Pro-Y with other amino acid residues resulted in no hydrolysis. The activity of the PIPase may depend on an active carboxyl group and on an active seryl residue but not on metal cations. Diethylpyrocarbonate inactivated the enzyme in a reaction that was not reversible upon addition of NH2OH. The enzyme contains a relatively large percentage (ca. 15%) of proline residues. The dominance of the PIPase activity among aminopeptidase activities present in T. denticola and the proposed location of the enzyme in the outer cell envelope suggest that it has a vital function in the propagation of the cells within their biological niche (inflamed human periodontal tissues). The biologic role of the PIPase may be envisaged as in the termination of the overall peptidolytic cascade (liberating free proline and other amino acids), whereby host tissue proteins and peptides are first processed and inactivated by other peptidases possibly present within the same confines as the PIPase.

Amino Acid Sequence↗
PubMed1996

Epitope mapping of B-cell determinants on the 15-kilodalton lipoprotein of Treponema pallidum (Tpp15) with synthetic peptides.

The antigenicity of the 15-kDa lipoprotein of Treponema pallidum (Tpp15 or TpN15) was comprehensively evaluated in epitope-scanning studies with overlapping deca- and octapeptides and polygonal rabbit and human infant immunoglobulins (Igs) and antisera. This approach enabled us to identify potentially important regions and to determine the optimal dilutions of Igs or antisera for use in further studies. IgM and IgG from both species were capable of recognizing multiple, continuous epitopes. A total of 13 peptides, principally clustered in the central regions of the protein, were recognized by all syphilitic sera and Ig fractions. On the basis of window analyses, frequency profiles, and alanine substitution studies, five heptapeptides were selected for mimetic studies. Two of these five immunodominant, continuous epitopes initially appeared to be species specific; however, antisera elicited against mimetics of all five epitopes were polyspecific, recognizing similar motifs on several other treponemal proteins, including those of avirulent organisms. The only mimetic which yielded positive reactions with infant IgM and syphilitic sera in the absence of cross-reactions with rabbit antisera to avirulent treponemes was the variant of the VMYASSG motif. These findings are relevant to the development of simple, inexpensive assays for the serodiagnosis of active syphilis.

Amino Acid Sequence↗
PubMed1996

The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.

The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.

Anions↗
PubMed1996

Activation of human monocytic cells by Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides proceeds via a pathway distinct from that of lipopolysaccharide but involves the transcriptional activator NF-kappa B.

There is increasing evidence that lipoproteins of Treponema pallidum and Borrelia burgdorferi are key inflammatory mediators during syphilis and Lyme disease. A principal objective of the present study was to identify more precisely similarities and divergences among lipopolysaccharide (LPS)- and lipoprotein-lipopeptide-induced immune cell signaling events. Like LPS, purified native B. burgdorferi OspA and synthetic analogs of OspA, OspB, and two T. pallidum lipoproteins (Tpp47 and Tpp17) all induced NF-kappa B translocation in THP-1 human monocytoid cells. Acylation of OspA and the synthetic peptides was requisite for cell activation. Polymyxin B abrogated only the response to LPS. By using 70Z/3-derived pre-B-cell lines either lacking or expressing human CD14 (the LPS receptor), it was observed that expression of human CD14 imparted responsiveness to LPS but not to OspA or spirochetal lipopeptides (assessed by induction of NF-kappa B and expression of surface immunoglobulin M). Finally, the biological relevance of the observation that T. pallidum lipoproteins-lipopeptides induce both NF-kappa B and cytokine production in monocytes was supported by the ability of the synthetic analogs to promote human immunodeficiency virus replication in chronically infected U1 monocytoid cells; these observations also suggest a potential mechanism whereby a syphilitic chancre can serve as a cofactor for human immunodeficiency virus transmission. The combined data lend additional support to the proposal that spirochetal lipoproteins and LPS initiate monocyte activation via different cell surface events but that the signaling pathways ultimately converge to produce qualitatively similar cellular responses.

Amino Acid Sequence↗
PubMed1997

Characterization of a methyl-accepting chemotaxis protein gene, dmcA, from the oral spirochete Treponema denticola.

A gene, dmcA, expressing a methyl-accepting chemotaxis protein (MCP) from the oral spirochete Treponema denticola has been characterized. The gene was initially identified as an open reading frame immediately upstream from the previously characterized prtB protease gene of strain ATCC 35405. Nucleotide sequencing of the dmcA gene revealed a potential 57-kDa protein product with extensive homology with the signaling regions of MCPs from a variety of bacteria. The protein expressed in Escherichia coli cross-reacted with anti-Trg (E. coli MCP) serum, confirming its homology with MCPs. Northern blot and primer extension analyses identified the transcription start site of the monocistronic dmcA mRNA. By utilizing a T. denticola gene inactivation system recently developed in this laboratory, a mutant defective in the dmcA gene, HL0501, was constructed. The mutant was demonstrated to be defective in chemotaxis toward nutrients. In addition, the methylation profiles of cellular proteins indicated altered MCPs in the mutant relative to those of the parental strain. These results indicate that we have identified an MCP gene in the oral spirochete which plays a significant role in the chemotactic response of the organism.

Amino Acid Sequence↗
PubMed1997

Identification of homologs for thioredoxin, peptidyl prolyl cis-trans isomerase, and glycerophosphodiester phosphodiesterase in outer membrane fractions from Treponema pallidum, the syphilis spirochete.

In this study, we characterized candidate rare outer membrane (OM) proteins with apparent molecular masses of 19, 27, 38, and 38.5 kDa, which had been identified previously in OM fractions from Treponema pallidum (J. D. Radolf et al., Infect. Immun. 63:4244-4252, 1995). Using N-terminal and internal amino acid sequences, a probe for the 19-kDa candidate was PCR amplified and used to screen a T. pallidum genomic library in Lambda Zap II. The corresponding gene (tlp) encoded a homolog for periplasmic thioredoxin-like proteins (Tlp), which reduce c-type cytochromes. A degenerate oligonucleotide derived from the N terminus of the 27-kDa protein was used to PCR amplify a duplex probe from a T. pallidum genomic library in pBluescript II SK+. With this probe, the corresponding gene (ppiB) was identified and found to code for a presumptive periplasmic cyclophilin B-type peptidyl prolyl cis-trans isomerase (PpiB). We postulate that PpiB assists the folding of proteins within the T. pallidum periplasmic space. The N terminus of the 38-kDa candidate was blocked to Edman degradation. However, internal sequence data revealed that it was basic membrane protein (Bmp), a previously characterized, signal peptidase I-processed protein. Triton X-114 phase partitioning revealed that despite its name, Bmp is hydrophilic and therefore likely to be periplasmic. The final candidate was also blocked to Edman degradation; as before, a duplex probe was PCR amplified with degenerate primers derived from internal sequences. The corresponding gene (glpQ) coded for a presumptively lipid-modified homolog of glycerophosphodiester phosphodiesterase (GlpQ). Based upon findings with other treponemal lipoproteins, the hydrophilic GlpQ polypeptide is thought to be anchored by N-terminal lipids to the periplasmic leaflet(s) of the cytoplasmic membrane and/or OM. The discovery of T. pallidum periplasmic proteins with potentially defined functions provides fresh insights into a poorly understood aspect of treponemal physiology. At the same time, however, these findings also raise important issues regarding the use of OM preparations for identifying rare OM proteins of T. pallidum.

Amino Acid Sequence↗
PubMed1997

gamma-Glutamyltransferase from the outer cell envelope of Treponema denticola ATCC 35405.

The human oral spirochete Treponema denticola ATCC 35405 was shown to exhibit relatively high enzyme activity toward the gamma-glutamyl amide bond present in N-gamma-L-glutamyl-4-nitroaniline. The enzyme responsible for this catalysis (gamma-glutamyltransferase [GGT]; EC 2.3.2.2) was purified by means of fast protein liquid chromatography to two sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-pure forms from a mild (0.1%) Triton X-100 extract of washed cells. The GGT was studied primarily with regard to its hydrolytic activity by using N-gamma-L-glutamyl-4-nitroaniline as a substrate, although the GGT was shown to catalyze transpeptidation reactions. The high-molecular-mass form of the GGT gave a value of about 213 kDa by SDS-PAGE when heat treatment was omitted and one of 26 kDa after heat treatment; mass spectrometry gave a value of 26.877. The larger form may represent an aggregate with nonprotein structures (possibly of a carbohydrate nature). The preliminary N-terminal sequence of the GGT is MKKPLIGITGSXLYETSQXXF. The enzyme was highly active on glutathione, transferring its Glu residue either to a water molecule or to the Gly-L-Leu dipeptide. The GGT stability was absolutely dependent on the presence of free thiol(s), while no evidence of metalloenzyme nature was obtained. The proposed location of the GGT in the outer cell envelope and its high activity on glutathione, a major nonprotein thiol present in virtually all cells, suggest that the GGT may play a role in the propagation of T. denticola within inflamed periodontal tissues.

Amino Acid Sequence↗
PubMed1997

Evidence for a methyl-accepting chemotaxis protein gene (mcp1) that encodes a putative sensory transducer in virulent Treponema pallidum.

The clinical and histopathological manifestations of syphilis and the invasive behavior of Treponema pallidum in tissue culture systems reflect the propensity for treponemes to migrate through skin, hematogenously disseminate, and invade targeted tissues. Treponemal motility is believed to be essential to this process and thereby an important facet of syphilis pathogenesis. By analogy with other bacterial pathogens, it is plausible that treponemal motility and tissue invasion are modulated by sensory transduction events associated with chemotactic responses. Recent studies have demonstrated the existence in T. pallidum of accessory molecules typically associated with sensory transduction events involving methyl-accepting chemotaxis proteins (MCPs). Intrinsic radiolabeling of T. pallidum in vitro with L-[methyl-3H] methionine revealed one methylated treponemal polypeptide with an apparent molecular mass of 64 kDa. A degenerate oligonucleotide probe corresponding to a highly conserved C-terminal domain within Bacillus subtilis and Escherichia coli MCPs was used in Southern blotting of T. pallidum DNA to identify and subsequently clone a putative T. pallidum MCP gene (mcp1). Computer analyses predicted a near-consensus promoter upstream of mcp1, and primer extension analysis employing T. pallidum RNA revealed a transcriptional initiation site. T. pallidum mcp1 encoded a 579-amino-acid (64.6-kDa) polypeptide which was highly homologous to at least 69 other known or putative sensory transducer proteins, with the highest degrees of homology existing between the C terminus of mcp1 and the C-terminal (signaling) domains of the other bacterial MCPs. Other salient features of Mcp1 included (i) six potential membrane-spanning domains at the N terminus, (ii) two predicted alpha-helical coiled coil regions containing at least three putative methylation sites, and (iii) homologies with two ligand-binding domains (LI-1 and LI-2) of the E. coli MCPs Trg and Tar. This study is the first to provide both metabolic and genetic evidence for an MCP sensory transducer in T. pallidum. The combined findings prompt key questions regarding the relationship(s) among sensory transduction, regulation of endoflagellar rotation, and chemotactic responses (in particular, the role of glucose) during virulence expression by T. pallidum.

Amino Acid Sequence↗
PubMed1997

Genetic and transcriptional analysis of flgB flagellar operon constituents in the oral spirochete Treponema denticola and their heterologous expression in enteric bacteria.

Oral spirochetes possess many potential virulence factors, including the capacity for tissue invasion and persistence despite a vigorous host immune response. In an attempt to identify treponemal immunoreactive components, sera derived from individuals with advanced periodontal disease were used as a reagent to isolate recombinant bacteriophage lambda clones expressing antigens of the oral spirochete Treponema denticola ATCC 35405. Nucleotide sequence analysis of a clone expressing three immunoreactive products has revealed seven T. denticola genes which appear to encode homologs of flagellar basal body constituents, FlgB, FlgC, FliE, and FliF, a flagellar switch component, FliG, and the putative flagellar export proteins, FliH and FliI, initially characterized in Salmonella typhimurium. Also identified was a gene resembling fliJ. Primer extension analysis identified a transcriptional start site 5' to the treponemal flgB gene. Appropriately spaced with respect to this start site was a sigma28 binding motif. The absence of additional identifiable sigma factor binding motifs within the treponemal sequence and the proximity of adjacent genes suggested operonic arrangement, and reverse transcriptase PCR provided evidence of cotranscription. Supporting the identification of these genes as flagellar components, heterologous expression in enteric bacteria of the putative switch basal body genes from T. denticola interfered with motility. Specifically, the presence of a plasmid expressing treponemal fliG reduced swarming motility in S. typhimurium, while in Escherichia coli, this plasmid conferred a nonmotile phenotype and a reduction in flagellar number. Thus, while spirochetal flagella are subject to unique synthetic and functional constraints, the organization of flagellar genes and the presence of sigma28-like elements are reminiscent of the flagellar systems of other bacteria, and there appears to be sufficient conservation of constituent proteins to allow interaction between T. denticola switch-basal body proteins and the flagellar machinery of gram-negative bacteria.

Amino Acid Sequence↗
PubMed1997

Activation of the interleukin-1beta precursor by Treponema denticola: a potential role in chronic inflammatory periodontal diseases.

There are several indications suggesting that interleukin-1beta (IL-1beta) may play an important role in inflammatory periodontal diseases. We hypothesized that periodontal sites would represent a unique combination of both cellular sources of IL-1beta precursor (pro-IL-1beta) and microbial proteases and proposed that Treponema denticola, a suspected periodontal pathogen, would play a critical role in the inflammatory nature of adult chronic periodontitis by activating pro-IL-1beta. The aim of this study was thus to demonstrate the proteolytic cleavage and activation of the inactive precursor pro-IL-1beta by T. denticola. After incubation of bacterial cells with recombinant pro-IL-1beta, proteolytic cleavage was monitored by Western immunoblotting, and the biological activity of the digestion products was tested in a bioassay. We report here that T. denticola can cleave pro-IL-1beta to yield two fragments with molecular masses of 18 and 19 kDa. Cleavage products showed a dose-dependent biological activity in the thymocyte proliferation bioassay, and this activity was inhibited by anti-IL-1beta neutralizing antibodies. These results suggest that T. denticola may have a proinflammatory role in periodontal diseases.

Animals↗
PubMed1997

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.

Cystathionine gamma-Lyase↗
PubMed1998

Function and protective capacity of Treponema pallidum subsp. pallidum glycerophosphodiester phosphodiesterase.

Infectious syphilis, caused by the spirochete bacterium Treponema pallidum subsp. pallidum, remains a public health concern worldwide. The immune-response evasion mechanisms employed by T. pallidum are poorly understood, and prior attempts to identify immunoprotective antigens for subsequent vaccine design have been unsuccessful. Previous investigations conducted in our laboratory identified the T. pallidum glycerophosphodiester phosphodiesterase as a potential immunoprotective antigen by using a differential immunologic expression library screen. In studies reported here, heterologous expression of the T. pallidum glycerophosphodiester phosphodiesterase in Escherichia coli yielded a full-length, enzymatically active protein. Characterization of the recombinant molecule showed it to be bifunctional, in that it exhibited specific binding to human immunoglobulin A (IgA), IgD, and IgG in addition to possessing enzymatic activity. IgG fractionation studies revealed specific binding of the recombinant enzyme to the Fc fragment of human IgG, a characteristic that may play a role in enabling the syphilis spirochete to evade the host immune response. In further investigations, immunization with the recombinant enzyme significantly protected rabbits from subsequent T. pallidum challenge, altering lesion development at the sites of challenge. In all cases, animals immunized with the recombinant molecule developed atypical pale, flat, slightly indurated, and nonulcerative reactions at the challenge sites that resolved before lesions appeared in the control animals. Although protection in the immunized rabbits was incomplete, as demonstrated by the presence of T. pallidum in the rabbit infectivity test, glycerophosphodiester phosphodiesterase nevertheless represents a significantly immunoprotective T. pallidum antigen and thus may be useful for inclusion in an antigen cocktail vaccine for syphilis.

Animals↗
PubMed1998

Filamentous actin disruption and diminished inositol phosphate response in gingival fibroblasts caused by Treponema denticola.

Previous reports have shown that Treponema denticola causes rearrangement of filamentous actin (F-actin) in human gingival fibroblasts (HGF). The purpose of this investigation was to determine the effect of T. denticola on the generation of inositol phosphates (IPs) in relation to a time course for F-actin disruption in HGF. Cultured HGF were exposed to washed cells of T. denticola ATCC 35405 for 140 min. Changes in the fluorescence intensity of rhodamine-phalloidin-labeled F-actin in serial optical sections of single HGF were quantified by confocal microscopy image analysis. The percentage of cells with stress fiber disruption was also determined by fluorescence microscopy. Challenge with T. denticola caused a significant reduction in F-actin within the first hour, especially at the expense of F-actin in the ventral third of the cells, and a significant increase in the percentage of HGF with altered stress fiber patterns. Significant concentration-dependent disruption of stress fibers was also caused by HGF exposure to a Triton X-100 extract of T. denticola outer membrane (OM). IPs were measured by a radiotracer assay based on the incorporation of myo-[3H]inositol into IPs in HGF incubated with LiCl to inhibit endogenous phosphatases. HGF challenge with several strains of T. denticola and the OM extract of T. denticola ATCC 35405 resulted in a diminished accumulation of radiolabeled IPs relative to both 15 and 1% fetal bovine serum, which served as strongly positive and background control agonists, respectively. The significantly diminished IP response to T. denticola ATCC 35405 occurred within 60 min, concomitant with significant reduction of total F-actin and disruption of stress fibers. Pretreatment with the proteinase inhibitor phenylmethylsulfonyl fluoride, which had previously been found to block T. denticola's degradation of endogenous fibronectin and detachment of HGF from the extracellular matrix, had little effect on F-actin stress fiber disruption and the IP response. Therefore, in addition to its major surface chymotrypsin-like properties, T. denticola expresses cytopathogenic activities that diminish the generation of IPs during the time course associated with significant cytoskeletal disruption in fibroblasts.

Actins↗
PubMed1998

Cytopathic effects of the major surface protein and the chymotrypsinlike protease of Treponema denticola.

Prominent antigens of Treponema denticola have been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of the T. denticola major surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.

Animals↗
PubMed1998

Molecular characterization of Treponema pallidum mcp2, a putative chemotaxis protein gene.

The nucleotide sequence of the Treponema pallidum mcp2 gene was determined. mcp2 encodes a 45.8-kDa protein whose deduced amino acid sequence has significant homology with the C-terminal region of bacterial methyl-accepting chemotaxis proteins (MCPs). The Mcp2 N terminus lacks the hydrophobic transmembrane regions present in most MCPs. An Mcp2 fusion protein was strongly reactive with antibody (HC23) to the highly conserved domain of MCPs and with rabbit syphilitic serum. Antibody HC23 reacted with six T. pallidum proteins, including a 45-kDa protein that may correspond to Mcp2. This protein was present in the aqueous phase from T. pallidum cells that were solubilized with Triton X-114 and phase partitioned.

Amino Acid Sequence↗
PubMed1999

Lack of humoral immune protection against Treponema denticola virulence in a murine model.

This study investigated the characteristics of humoral immune responses to Treponema denticola following primary infection, reinfection, and active immunization, as well as immune protection in mice. Primary infection with T. denticola induced a significant (400-fold) serum immunoglobulin G (IgG) response compared to that in control uninfected mice. The IgG response to reinfection was 20, 000-fold higher than that for control mice and 10-fold higher than that for primary infection. Mice actively immunized with formalin-killed treponemes developed serum antibody levels seven- to eightfold greater than those in animals after primary infection. Nevertheless, mice with this acquired antibody following primary infection or active immunization demonstrated no significant alterations of lesion induction or decreased size of the abscesses following a challenge infection. Mice with primary infection developed increased levels of IgG3, IgG2b, and IgG2a antibodies, with IgG1 being lower than the other subclasses. Reinfected mice developed enhanced IgG2b, IgG2a, and IgG3 and less IgG1. In contrast, immunized mice developed higher IgG1 and lower IgG3 antibody responses to infection. These IgG subclass distributions indicate a stimulation of both Th1 and Th2 activities in development of the humoral immune response to infection and immunization. Our findings also demonstrated a broad antigen reactivity of the serum antibody, which was significantly increased with reinfection and active immunization. Furthermore, serum antibody was effective in vitro in immobilizing and clumping the bacteria but did not inhibit growth or passively prevent the treponemal infection. These observations suggest that humoral immune responses, as manifested by antibody levels, isotype, and antigenic specificity, were not capable of resolving a T. denticola infection.

Animals↗
PubMed1999

Characterization of a novel methyl-accepting chemotaxis gene, dmcB, from the oral spirochete Treponema denticola.

Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named dmcB. Northern blot analysis suggested that dmcB was part of an operon structure containing prtB. Insertional inactivation of dmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of the dmcB gene also attenuated the methylation of the DmcA protein. These results suggest that the dmcB gene codes for an MCP in T. denticola which may interact with other MCPs in these organisms.

Amino Acid Sequence↗