Disruption of a type II endonuclease (TDE0911) enables Treponema denticola ATCC 35405 to accept an unmethylated shuttle vector

The oral spirochete Treponema denticola is associated with human periodontal disease. T. denticola ATCC 35405 and ATCC 33520 are two routinely used laboratory strains. Compared to T. denticola ATCC 33520, ATCC 35405 is more virulent but less accessible to genetic manipulations. For instance, the shuttle vectors of ATCC 33520 cannot be transformed into strain ATCC 35405. The lack of a shuttle vector has been a barrier to study the biology and virulence of T. denticola ATCC 35405. In this report, we hypothesize that T. denticola ATCC 35405 may have a unique DNA restriction-modification (R-M) system that prevents it from accepting the shuttle vectors of ATCC 33520 (e.g., the shuttle plasmid pBFC). To test this hypothesis, DNA restriction digestion, PCR, and Southern blot analyses were conducted to identify the differences between the R-M systems of these two strains. DNA restriction digestion analysis of these strains showed that only the cell extract from ATCC 35405 was able to digest pBFC. Consistently, PCR and Southern blot analyses revealed that the genome of T. denticola ATCC 35405 encodes three type II endonucleases that are absent in ATCC 33520. Among these three endonucleases, TDE0911 was predicted to cleave unmethylated double-stranded DNA and to be most likely responsible for the cleavage of unmethylated pBFC. In agreement with this prediction, the mutant of TDE0911 failed to cleave unmethylated pBFC plasmid, and it could accept the unmethylated shuttle vector. The study described here provides us with a new tool and strategy to genetically manipulate T. denticola, in particular ATCC 35405, and other strains that may carry similar endonucleases.     

A 52-kDa leucyl aminopeptidase from treponema denticola is a cysteinylglycinase that mediates the second step of glutathione metabolism

The metabolism of glutathione by the periodontal pathogen Treponema denticola produces hydrogen sulfide, which may play a role in the host tissue destruction seen in periodontitis. H2S production in this organism has been proposed to occur via a three enzyme pathway, gamma-glutamyltransferase, cysteinylglycinase (CGase), and cystalysin. In this study, we describe the purification and characterization of T. denticola CGase. Standard approaches were used to purify a 52-kDa CGase activity from T. denticola, and high pressure liquid chromatography electrospray ionization tandem mass spectrometry analysis of this molecule showed that it matches the amino acid sequence of a predicted 52-kDa protein in the T. denticola genome data base. A recombinant version of this protein was overexpressed in and purified from Escherichia coli and shown to catalyze the hydrolysis of cysteinylglycine (Cys-Gly) with the same kinetics as the native protein. Surprisingly, because sequence homology indicates that this protein is a member of a family of metalloproteases called M17 leucine aminopeptidases, the preferred substrate for the T. denticola protein is Cys-Gly (k cat/Km of 8.2 microm(-1) min(-1)) not l-Leu-p-NA (k cat/Km of 1.1 microm(-1) min(-1)). The activity of CGase for Cys-Gly is optimum at pH 7.3 and is enhanced by Mn2+, Co2+, or Mg2+ but not by Zn2+ or Ca2+. Importantly, in combination with the two other previously purified T. denticola enzymes, gamma-glutamyltransferase and cystalysin, CGase mediates the in vitro degradation of glutathione into the expected end products, including H2S. These results prove that T. denticola contains the entire three-step pathway to produce H2S from glutathione, which may be important for pathogenesis.     

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.     

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism.     

Molecular pathogenesis of the cell surface proteins and lipids from Treponema denticola

Treponema denticola, frequently isolated from the human oral cavity, is thought to be a major pathogen of human periodontal disease. Recent developments in molecular analysis have clarified the surface structure of this microorganism and the characteristics of its pathogenic factors. Structural analysis of the outer sheath showed T. denticola to have a new type of outer membrane lipid. Limited exposure of the major outer sheath protein is suggested by electron-microscopic analysis. A protease-deficient mutant has revealed the roles of the protease in the organization of the outer sheath material and in T. denticola pathogenicity. The surface features that contribute to the pathogenicity of T. denticola in periodontal disease are gradually being elucidated, and are reviewed.     

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.     

Conservation of msp, the gene encoding the major outer membrane protein of oral Treponema spp.

The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases. Previous studies showed that the Msp of T. denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane. The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J. C. Fenno, K.-H. Müller, and B. C. McBride, J. Bacteriol. 178:2489-2496, 1996). In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes. A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested. Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T. denticola ATCC 35404 and T. vincentii, reacted very weakly with the Msp protein of T. denticola ATCC 33520, and did not react with T. denticola OTK, T. socranskii, and T. pectinovorum. The msp loci of the T. denticola strains and T. vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405. Southern blot analysis showed at least three groups of related msp DNA sequences. Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50%. The entire msp DNA sequences of T. denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405. The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains. Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes.     

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.     

A series of new E. coli–Thermococcus shuttle vectors compatible with previously existing vectors

Hyperthermophilic microorganisms are an important asset in the toolkits of biotechnologists, biochemists and evolutionary biologists. The anaerobic archaeon, Thermococcus kodakarensis, has become one of the most useful hyperthermophilic model species, not least due to its natural competence and genetic tractability. Despite this, the range of genetic tools available for T. kodakarensis remains limited. Using sequencing and phylogenetic analyses, we determined that the rolling-circle replication origin of the cryptic mini-plasmid pTP2 from T. prieurii is suitable for plasmid replication in T. kodakarensis. Based on this replication origin, we present a novel series of replicative E. coli–T. kodakarensis shuttle vectors. These shuttle vectors have been constructed with three different selectable markers, allowing selection in a range of T. kodakarensis backgrounds. Moreover, these pTP2-derived plasmids are compatible with the single-existing E. coli–T. kodakarensis shuttle vector, pLC70. We show that both pTP2-derived and pLC70-derived plasmids replicate faithfully while cohabitating in T. kodakarensis cells. These plasmids open the door for new areas of research in plasmid segregation, DNA replication and gene expression.

     

The influence of cholesterol, progesterone, 4-androstenedione and testosterone on the growth of Treponema denticola ATCC 33520 in batch cultures

Previously, we have shown that reference and freshly isolated Treponema denticola cultures are capable of metabolising cholesterol, progesterone, 4-androstenedione and testosterone by means of 5alpha-reductase, 3beta-and 17beta-hydroxysteroid dehydrogenase activity [Clark DT, Soory M. The metabolism of cholesterol and certain hormonal steroids by Treponema denticola. Steroids. 2006;71:352-63. ]. The aim of the work presented in this paper was to investigate the modulation of T. denticola growth in batch cultures by these steroids, using T. denticola ATCC 33520 as a model system. Growth curves were summarised using statistics based on optical density and protein yield. Cholesterol was found to stimulate growth at concentrations of 10 and 25microg/mL. Certain hormonal steroids inhibited the maximum achievable optical density at concentrations of 1 and 10microg/mL while the minimum concentration shown to inhibit protein yield was 0.001microg/mL of progesterone. The potential of the hormonal steroids to inhibit growth was in the order of progesterone, 4-androstenedione and testosterone.     

Treponema denticola lipooligosaccharide activates gingival fibroblasts and upregulates inflammatory mediator production

In response to bacterial challenges, fibroblasts, a major constituent of gingival connective tissue, can produce immunoregulatory cytokines and proteolytic enzymes that may contribute to tissue destruction and the progression of periodontitis, a chronic inflammatory disease affecting tooth-supporting tissues, including alveolar bone. The spirochete Treponema denticola is a major etiological agent of periodontitis and can invade oral tissues. The aim of the present study was to investigate the inflammatory response of gingival fibroblasts to T. denticola lipooligosaccharide (LOS). T. denticola LOS induced significant production of various inflammatory mediators by fibroblasts, including interleukin-6, interleukin-8, monocyte chemoattractant protein 1, nitric oxide, and prostaglandin E(2). In addition, the secretion of matrix metalloproteinase 3, an enzyme active on basement membrane components, was also significantly increased. The response of fibroblasts was dose-dependent and much stronger following a 24 h stimulation period. The expression and/or phosphorylation state of several signaling proteins, including Fos, MKK1, MKK2, MKK3/6, NF-kappaB p50, and NF-kappaB p65, was enhanced following stimulation of fibroblasts with T. denticola LOS. In summary, T. denticola LOS induced an inflammatory response in gingival fibroblasts and may thus contribute to the immunopathogenesis of periodontitis and the progression of the disease.     

Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.     

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.     

Characterization, cloning, curing, and distribution in lactic acid bacteria of pLP1, a plasmid from Lactobacillus plantarum CCM 1904 and its use in shuttle vector construction

A small 2.1-kb plasmid called pLP1 was extracted from Lactobacillus plantarum CCM 1904 (ATCC 8014) and cloned into the Escherichia coli pUC19 plasmid. As determined by DNA-DNA Southern hybridization with a pLP1-radioactively labeled probe, other lactic acid bacteria such as L. curvatus, L. sake, Carnobacterium, and Leuconostoc mesenteroides harbor pLP1-related plasmids. Shuttle vectors based on the pLP1 replicon were constructed by inserting the erythromycin-resistance gene from pVA891 into the various pUC19-pLP1 constructions. pLP1-based shuttle vector transformation efficiencies (TE) by electroporation were compared to TE of a broad-host-range plasmid pGK12 in different lactobacilli strains. Expression of the pUC19-pLP1 plasmids in Escherichia coli maxicells showed that pLP1 encodes for a 37,000 MW protein which can act in trans allowing the replication of plasmids in which this protein is truncated. The pLP1-based shuttle vectors producing this protein replicate in lactobacilli and also in Bacillus subtilis. A pLP1-free strain was obtained by incompatibility with a pLP1-based shuttle vector introduced in L. plantarum CCM 1904 by electroporation. The absence of pLP1 has no incidence on the strain phenotype suggesting that pLP1 is not essential for the strain in our laboratory conditions.     

Gene inactivation in the oral spirochete Treponema denticola: construction of an flgE mutant.

Treponema denticola is implicated in the etiology of periodontal diseases. We now report the construction of a specific flgE mutant of T. denticola ATCC 35405 following electroporation utilizing an erythromycin resistance cassette inserted into an flgE DNA fragment. The resulting mutant displays no visible motility and lacks periplasmic flagella as would be predicted from inactivation of the gene for the flagellar hook protein.     

Treponema denticola induces interleukin-8 and macrophage chemoattractant protein 1 production in human umbilical vein epithelial cells

Treponema denticola, a major pathogen of periodontitis, has also been detected in the lesions of atherosclerosis. The aim of this study was to investigate induction of chemokine production in human umbilical vein endothelial cells (HUVECs) by T. denticola and determine whether those chemokines were degraded by a protease, dentilisin. T. denticola ATCC35405 or dentilisin-deficient mutant K1 were added to HUVECs and levels of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in the culture supernatants were determined by enzyme-linked immunosorbent assay. T. denticola ATCC35405 induced production of IL-8 in a time-dependent manner, with both production of IL-8 and expression of IL-8 mRNA showing higher levels than with exposure to dentilisin-deficient mutant K1. Although exposure to ATCC35405 induced expression of MCP-1 mRNA in the HUVECs, MCP-1 levels were remained similar to that in unstimulated cells. IL-8 and MCP-1 showed partial hydrolysis with exposure to T. denticola ATCC35405, but not with T. denticola K1. These results suggest that T. denticola can evade host defense mechanisms by modulating production of IL-8 and MCP-1, and that this play a role in the development of chronic infections such as periodontitis. The association of T. denticola infection to atherosclerosis was also discussed based on the present study.     

Transfer of conjugal elements in oral black-pigmented Bacteroides (Prevotella) spp. involves DNA rearrangements.

Conjugal genetic elements in isolates of oral black-pigmented Bacteroides denticola (Prevotella denticola) and B. intermedius (P. intermedia) transfer tetracycline and penicillin resistance in the absence of plasmids. Transverse alternating-field electrophoresis of restricted chromosomal DNAs from transconjugants revealed arrangements indicating that transfer and insertion can involve more than one 60-kb copy of the elements and occurs at strongly preferred sites in the recipient chromosome.     

Molecular cloning of the intronless EJ ras oncogene using a murine retrovirus shuttle vector

We have inserted a genomic clone of the human EJ bladder oncogene into a murine retrovirus shuttle vector. Cotransfection of this shuttle vector DNA containing the activated ras oncogene with molecularly cloned Moloney murine leukemia virus into NIH/3T3 cells was able to rescue a replicating and transforming retrovirus. The viral DNA from infected cells was excised by fusion to mouse COP-5 cells and recloned into Escherichia coli as plasmid DNA. Analysis of the recloned plasmids by size, restriction enzyme mapping, and DNA sequence indicated that approximately 5% of the recloned plasmids contained the intronless EJ ras oncogene.     

Development of Thermus-Escherichia shuttle vectors and their use for expression of the Clostridium thermocellum celA gene in Thermus thermophilus.

We describe the self-selection of replication origins of undescribed cryptic plasmids from Thermus aquaticus Y-VII-51B (ATCC 25105) and a Thermus sp. strain (ATCC 27737) by random insertion of a thermostable kanamycin adenyltransferase cartridge. Once selected, these autonomous replication origins were cloned into the Escherichia coli vector pUC9 or pUC19. The bifunctional plasmids were analyzed for their sizes, relationships, and properties as shuttle vectors for Thermus-Escherichia cloning. Seven different vectors with diverse kanamycin resistance levels, stabilities, transformation efficiencies, and copy numbers were obtained. As a general rule, those from T. aquaticus (pLU1 to pLU4) were more stable than those from the Thermus sp. (pMY1 to pMY3). To probe their usefulness, we used one of the plasmids (pMY1) to clone in E. coli a modified form of the cellulase gene (celA) from Clostridium thermocellum in which the native signal peptide was replaced in vitro by that from the S-layer gene of T. thermophilus HB8. The hybrid product was expressed and exported by E. coli. When the gene was transferred by transformation into T. thermophilus, the cellulase protein was also expressed and secreted at 70 degrees C.     

The metabolism of cholesterol and certain hormonal steroids by Treponema denticola

The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5alpha-reduce and further metabolise testosterone, 4-androstenedione, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography-mass spectroscopy. All the substrates were 5alpha-reduced. Both reference cultures and fresh isolates of T. denticola presented 3beta- and 17beta-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.