Treponema denticola TroR is a manganese- and iron-dependent transcriptional repressor

Treponema denticola harbours a genetic locus with significant homology to most of the previously characterized Treponema pallidum tro operon. Within this locus are five genes ( troABCDR ) encoding for the components of an ATP-binding cassette cation-transport system ( troABCD ) and a DtxR-like transcriptional regulator ( troR ). In addition, a σ 70 -like promoter and an 18 bp region of dyad symmetry were identified upstream of the troA start codon. This putative operator sequence demonstrated similarity to the T. pallidum TroR (TroR Tp ) binding sequence; however, the position of this motif with respect to the predicted tro promoters differed. Interestingly, unlike the T. pallidum orthologue, T. denticola TroR (TroR Td ) possesses a C-terminal Src homology 3-like domain commonly associated with DtxR family members. In the present study, we show that TroR Td is a manganese- and iron-dependent transcriptional repressor using Escherichia coli reporter constructs and in T. denticola . In addition, we demonstrate that although TroR Td possessing various C-terminal deletions maintain metal-sensing capacities, these truncated proteins exhibit reduced repressor activities in comparison with full-length TroR Td . Based upon these findings, we propose that TroR Td represents a novel member of the DtxR family of transcriptional regulators and is likely to play an important role in regulating both manganese and iron homeostases in this spirochaete.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72265/1/j.1365-2958.2008.06418.x.pd

Expression of Treponema denticola Oligopeptidase B in Escherichia coli

Treponema denticola is a small anaerobic spirochete often isolated from periodontal lesions and closely associated with periodontal diseases. This bacterium possesses a particular arginine peptidase activity (previously called “BANA-peptidase” or “trypsin-like enzyme”) that is common to the three cultivable bacterial species most highly associated with severe periodontal disease. We recently reported the identification of the opdB locus that encodes the BANA-peptidase activity of T. denticola through DNA sequencing and mutagenesis studies. In the present study, we report expression of T. denticola OpdB peptidase in Escherichia coli. The opdB PCR product was cloned into pET30b and then transformed into the E. coli BL21 (DE3)/pLysS expression strain. Assays of enzymatic activities in E. coli containing T. denticola opdB showed BANA-peptidase activity similar to that of T. denticola. Availability of this recombinant expression system producing active peptidase will facilitate characterization of the potential role of this peptidase in periodontal disease etiology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41338/1/284_2003_Article_4168.pd

Treponema denticola increases MMP‐2 expression and activation in the periodontium via reversible DNA and histone modifications

Host‐derived matrix metalloproteinases (MMPs) and bacterial proteases mediate destruction of extracellular matrices and supporting alveolar bone in periodontitis. The Treponema denticola dentilisin protease induces MMP‐2 expression and activation in periodontal ligament (PDL) cells, and dentilisin‐mediated activation of pro‐MMP‐2 is required for cellular fibronectin degradation. Here, we report that T. denticola regulates MMP‐2 expression through epigenetic modifications in the periodontium. PDL cells were treated with epigenetic enzyme inhibitors before or after T. denticola challenge. Fibronectin fragmentation, MMP‐2 expression, and activation were assessed by immunoblot, zymography, and qRT‐PCR, respectively. Chromatin modification enzyme expression in T. denticola‐challenged PDL cells and periodontal tissues were evaluated using gene arrays. Several classes of epigenetic enzymes showed significant alterations in transcription in diseased tissue and T. denticola‐challenged PDL cells. T. denticola‐mediated MMP‐2 expression and activation were significantly reduced in PDL cells treated with inhibitors of aurora kinases and histone deacetylases. In contrast, DNA methyltransferase inhibitors had little effect, and inhibitors of histone acetyltransferases, methyltransferases, and demethylases exacerbated T. denticola‐mediated MMP‐2 expression and activation. Chronic epigenetic changes in periodontal tissues mediated by T. denticola or other oral microbes may contribute to the limited success of conventional treatment of chronic periodontitis and may be amenable to therapeutic reversal.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142926/1/cmi12815.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142926/2/cmi12815_am.pd

Characterization of Treponema denticola pyrF encoding orotidine-5′-monophosphate decarboxylase

The Treponema denticola ATCC 35405 genome annotation contains most of the genes for de novo pyrimidine biosynthesis. To initiate characterization of pyrimidine synthesis in Treponema , we focused on TDE2110 (the putative pyrF , encoding orotidine-5′-monophosphate decarboxlyase). Unlike the parent strain, an isogenic pyrF mutant was resistant to 5-fluoroorotic acid. In complex medium, growth of the pyrF mutant was independent of added uracil, indicating activity of a uracil uptake/salvage pathway. Transcription of pyrF was greatly reduced in T. denticola grown in excess uracil, demonstrating that de novo pyrimidine synthesis is regulated and suggesting a feedback mechanism. Treponema denticola PyrF complemented uracil auxotrophy in an Escherichia coli pyrF mutant. This study provides biochemical confirmation of T. denticola genome predictions of de novo and salvage pyrimidine pathways and provides proof of concept that pyrF has potential as a selectable marker in T. denticola .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75261/1/j.1574-6968.2006.00589.x.pd

The antimicrobial efficacy of ‘MGP’ gutta-percha in vitro

Shur AL, Sedgley CM, Fenno JC. The antimicrobial efficacy of ‘MGP’ gutta-percha in vitro . International Endodontic Journal , 36 , 616–621, 2003. Aim  To determine whether ‘MGP’ gutta-percha (Westport, CT, USA), a commercially available gutta-percha containing iodoform, inhibits the growth of potential endodontic pathogens. Methodology  Inocula of Enterococcus faecalis , Staphylococcus aureus , Escherichia coli , Pseudomonas aeruginosa , Streptococcus sanguis , Fusobacterium nucleatum and Actinomyces odontolyticus were spread onto the surface of agar plates. ‘MGP’ gutta-percha cones presoaked in sterile water were transferred to the inoculated agar and incubated at 37 °C aerobically or anaerobically as required for optimal growth. Identical studies were performed using iodoform-free gutta-percha and sterile paper disks saturated with 10% povidone–iodine. Following incubation, zones of inhibition around the ‘MGP’ gutta-percha, iodoform-free gutta-percha and disks were evaluated. Results  Povidone–iodine inhibited all the strains. Iodoform-free gutta-percha inhibited S. sanguis and A. odontolyticus . ‘MGP’ gutta-percha inhibited S. aureus , S. sanguis , A. odontolyticus and F. nucleatum . Neither iodoform-free gutta-percha nor ‘MGP’ gutta-percha inhibited growth of E. faecalis , E. coli or P. aeruginosa . Conclusions  Compared to iodoform-free gutta-percha, iodoform-containing ‘MGP’ gutta-percha had an inhibitory effect in vitro on S. aureus and F. nucleatum , but not on E. faecalis , E. coli or P. aeruginosa .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75637/1/j.1365-2591.2003.00699.x.pd

Composition and Localization of Treponema denticola Outer Membrane Complexes ▿

The Treponema denticola outer membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated that prcB, located upstream of and cotranscribed with prcA and prtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of both prcA and prtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-high-molecular-weight outer membrane complex

The Chymotrypsin-Like Protease Complex of Treponema denticola ATCC 35405 Mediates Fibrinogen Adherence and Degradation▿

Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aα and Bβ chains of fibrinogen, but not the γ chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, ∼100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100°C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP− mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP−) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP

Analysis of a Unique Interaction between the Complement Regulatory Protein Factor H and the Periodontal Pathogen Treponema denticola▿

Treponema denticola, a spirochete associated with periodontitis, is abundant at the leading edge of subgingival plaque, where it interacts with gingival epithelia. T. denticola produces a number of virulence factors, including dentilisin, a protease which is cytopathic to host cells, and FhbB, a unique T. denticola lipoprotein that binds complement regulatory proteins. Earlier analyses suggested that FhbB specifically bound to factor H (FH)-like protein 1 (FHL-1). However, by using dentilisin-deficient mutants of T. denticola, we found that T. denticola preferentially binds FH and not FHL-1, and that FH is then cleaved by dentilisin to yield an FH subfragment of ∼50 kDa. FH bound to dentilisin-deficient mutants but was not cleaved and retained its ability to serve as a cofactor for factor I in the cleavage of C3b. To assess the molecular basis of the interaction of FhbB with FH, mutational analyses were conducted. Replacement of specific residues in widely separated domains of FhbB and disruption of a central alpha helix with coiled-coil formation probability attenuated or eliminated FH binding. The data presented here are the first to demonstrate the retention at the cell surface of a proteolytic cleavage product of FH. The precise role of this FH fragment in the host-pathogen interaction remains to be determined