Novel transcriptional regulator OxtR1 regulates potential ferrodoxin in response to oxygen stress in Treponema denticola.

OBJECTIVE: Treponema denticola has been strongly implicated in the pathogenesis of chronic periodontitis. Previously, we reported that the potential transcriptional regulator TDE_0259 (oxtR1) is upregulated in the bacteriocin ABC transporter gene-deficient mutant. OxtR1 may regulate genes to adapt to environmental conditions during colonization; however, the exact role of the gene in T. denticola has not been reported. Therefore, we investigated its function using an oxtR1-deficient mutant. METHODS: The growth rates of the wild-type and oxtR1 mutant were monitored under anaerobic conditions; their antibacterial agent susceptibility and gene expression were assessed using a liquid dilution assay and DNA microarray, respectively. An electrophoretic mobility shift assay was performed to investigate the binding of OxtR1 to promoter regions. RESULTS: The growth rate of the bacterium was accelerated by the inactivation of oxtR1, and the mutant exhibited an increased minimum inhibitory concentration against ofloxacin. We observed a relative increase in the expression of genes associated with potential ferrodoxin (TDE_0260), flavodoxin, ABC transporters, heat-shock proteins, DNA helicase, iron compounds, and lipoproteins in the mutant. OxtR1 expression increased upon oxygen exposure, and oxtR1 complementation suppressed the expression of potential ferrodoxin. Our findings also suggested that OxtR1 binds to a potential promoter region of the TDE_0259-260 operon. Moreover, the mutant showed a marginal yet significantly faster growth rate than the wild-type strain under H2O2 exposure. CONCLUSION: The oxygen-sensing regulator OxtR1 plays a role in regulating the expression of a potential ferrodoxin, which may contribute to the response of T. denticola to oxygen-induced stress.

Transcriptional and functional characterizations of multiple flagellin genes in spirochetes.

The flagellar filament is a helical propeller for bacterial locomotion. In external flagellates, the filaments are mostly homopolymers of a single flagellin protein. By contrast, the flagellar filaments of spirochetes are mostly heteropolymers of multiple flagellin proteins. This report seeks to investigate the role of multiple flagellin proteins using the oral spirochete Treponema denticola as a model. First, biochemical and genetic studies uncover that the flagellar filaments of T. denticola mainly comprise four proteins, FlaA, FlaB1, FlaB2, and FlaB3, in a defined stoichiometry. Second, transcriptional analyses reveal that the genes encoding these four proteins are regulated by two different transcriptional factors, sigma28 and sigma70 . Third, loss-of-function studies demonstrate that each individual flagellin protein contributes to spirochete motility, but none of them is absolutely required. Last, we provide genetic and structural evidence that FlaA forms a "seam"-like structure around the core and that deletion of individual flagellin protein alters the flagellar homeostasis. Collectively, these results demonstrate that T. denticola has evolved a unique mechanism to finely regulate its flagellar filament gene expression and assembly which renders the organelle with the right number, shape, strength, and structure for its distinct motility.

Effect of Treponema Denticola Infection on Epithelial Cells.

Chronic periodontitis is an infectious disease caused by periodontopathic bacteria in subgingival plaque. One major pathogen of this disease, Treponema denticola, has several virulence factors, including a major surface protein (Msp) and the surface protease dentilisin. The cytopathic effects of periodontopathic bacteria on epithelial cells disrupt the integrity of the barrier junction, resulting in the inflammation of periodontal tissue. The aim of this study was to investigate the effect of T. denticola virulence factors dentilisin and Msp on epithelial cells. The effects of T. denticola wild-type, Msp-mutant, and dentilisin-mutant strains on the contact junction in Madin-Darby canine kidney epithelial cells was evaluated based on ohmic values. Cultured oral carcinoma epithelial cells were scratched and exposed to the selected T. denticola strains and cell migration determined. Subsequent degradation of adherence proteins and proteins in the contact junctions was evaluated. Dissociation of cell contact junctions was detected in cells infected with wild-type T. denticola approximately 30 min after infection, but not in those exposed to the mutants. Inhibition of migration was observed in the wild-type and Msp-deficient mutants. The adherent proteins focal adhesion kinase, ZO-1, and paxillin were hydrolyzed by infection with the wild-type and Msp mutants. These results indicate that T. denticola disrupts the function of epithelial cells by hydrolyzing proteins at the intercellular junction and inhibiting healing of epithelial cells via hydrolyzed proteins associated with focal adhesion; Msp was also associated with these effects.

Inhibitory effect of Streptococcus salivarius K12 and M18 on halitosis in vitro.

BACKGROUND: The aim of the study was to observe the antimicrobial activity of Porphyromonas gingivalis and Treponema denticola as well as the effect on reducing volatile sulfur compounds (VSCs). MATERIALS AND METHODS: After P. gingivalis and T. denticola were cultured with or without Streptococcus salivarius K12 and M18, VSCs were measured by Oral Chroma. In order to analyze the mechanism for malodor control, the antimicrobial activity of S. salivarius K12 and M18 against P. gingivalis and T. denticola was assessed. SPSS 21.0 was used for data analysis with the Kruskal-Wallis and Jonckheere-Terpstra tests. Mann-Whitney test was applied for post hoc analysis. RESULTS: P. gingivalis and T. denticola VSC levels were reduced by high concentrations of S. salivarius K12 and M18 during coculture. The concentrations were lower than those of single culture (p < .05). An antimicrobial effect was detected on P. gingivalis, and T. denticola by 50% S. salivarius K12 and M18. The spent culture medium and whole bacteria of S. salivarius K12 and M18 reduced the levels of VSCs below the amount in a single culture of P. gingivalis and T. denticola (p < .05). CONCLUSION: S. salivarius K12 and M18 decreased the levels of VSCs originating from P. gingivalis and T. denticola.

Glutathione catabolism by Treponema denticola impacts its pathogenic potential.

Treponema denticola is a spirochete that is etiologic for periodontal diseases. This bacterium is one of two periodontal pathogens that have been shown to have a complete three step enzymatic pathway (GTSP) that catabolizes glutathione to H2S. This pathway may contribute to the tissue pathology seen in periodontitis since diseased periodontal pockets have lower glutathione levels than healthy sites with a concomitant increase in H2S concentration. In order to be able to demonstrate that glutathione catabolism by the GTSP is critical for the pathogenic potential of T. denticola, allelic replacement mutagenesis was used to make a deletion mutant (Δggt) in the gene encoding the first enzyme in the GTSP. The mutant cannot produce H2S from glutathione since it lacks gamma-glutamyltransferase (GGT) activity. The hemolytic and hemoxidation activities of wild type T. denticola plus glutathione are reduced to background levels with the Δggt mutant and the mutant has lost the ability to grow aerobically when incubated with glutathione. The Δggt bacteria with glutathione cause less cell death in human gingival fibroblasts (hGFs) in vitro than do wild type T. denticola and the levels of hGF death correlate with the amounts of H2S produced. Importantly, the mutant spirochetes plus glutathione make significantly smaller lesions than wild type bacteria plus glutathione in a mouse back lesion model that assesses soft tissue destruction, a major symptom of periodontal diseases. Our results are the first to prove that T. denticola thiol-compound catabolism by its gamma-glutamyltransferase can play a significant role in the in the types of host tissue damage seen in periodontitis.

The Msp Protein of Treponema denticola Interrupts Activity of Phosphoinositide Processing in Neutrophils.

Periodontal disease is a significant health burden, causing tooth loss and poor oral and overall systemic health. Dysbiosis of the oral biofilm and a dysfunctional immune response drive chronic inflammation, causing destruction of soft tissue and alveolar bone supporting the teeth. Treponema denticola, a spirochete abundant in the plaque biofilm of patients with severe periodontal disease, perturbs neutrophil function by modulating appropriate phosphoinositide (PIP) signaling. Through a series of immunoblotting and quantitative PCR (qPCR) experiments, we show that Msp does not alter the gene transcription or protein content of key enzymes responsible for PIP3 signaling: 3' phosphatase and tensin homolog (PTEN), phosphatidylinositol 3-kinase (PI3K), or 5' Src homology 2 domain-containing inositol phosphatase 1 (SHIP1). Instead, using immunoblotting and enzyme-linked immunosorbent assays (ELISAs), we found that Msp activates PTEN through dephosphorylation specifically at the S380 site. Msp in intact organisms or outer membrane vesicles also restricts PIP signaling. SHIP1 phosphatase release was assessed using chemical inhibition and immunoprecipitation to show that Msp moderately decreases SHIP1 activity. Msp also prevents secondary activation of the PTEN/PI3K response. We speculate that this result is due to the redirection of the PIP3 substrate away from SHIP1 to PTEN. Immunofluorescence microscopy revealed a redistribution of PTEN from the cytoplasm to the plasma membrane following exposure to Msp, which may contribute to PTEN activation. Mechanisms of how T. denticola modulates and evades the host immune response are still poorly described, and here we provide further mechanistic evidence of how spirochetes modify PIP signaling to dampen neutrophil function. Understanding how oral bacteria evade the immune response to perpetuate the cycle of inflammation and infection is critical for combating periodontal disease to improve overall health outcomes.

Treponema denticola Induces Epithelial Barrier Dysfunction in Polarized Epithelial Cells.

Treponema denticola, an anaerobic spirochete found mainly in the oral cavity, is associated with periodontal disease and has a variety of virulence factors. Although in vitro studies have shown that T. denticola is able to penetrate epithelial cell monolayers, its effect on the epithelial barrier junction is not known. Human gingival epithelial cells are closely associated with adjacent membranes, forming barriers in the presence of tight junction proteins, including zonula occludens-1 (ZO-1), claudin-1, and occludin. Tight junction proteins are also expressed by Madin-Darby canine kidney (MDCK) cells in culture. In this study, the MDCK cell profile was investigated following infection with T. denticola (ATCC 35405) wild-type, as well as with its dentilisin-deficient mutant, K1. Basolateral exposure of MDCK cell monolayers to T. denticola at a multiplicity of infection (MOI) of 104 resulted in a decrease in transepithelial electrical resistance (TER). Transepithelial electrical resistance in MDCK cell monolayers also decreased following apical exposure to T. denticola (MOI=104), although this took longer with basolateral exposure. The effect on the TER was time-dependent and required the presence of live bacteria. Meanwhile, MDCK cell viability showed a decrease with either basolateral or apical exposure. Immunofluorescence analysis demonstrated decreases in the amounts of immunoreactive ZO-1 and claudin-1 in association with disruption of cell-cell junctions in MDCK cells exposed apically or basolaterally to T. denticola. Western blot analysis demonstrated degradation of ZO-1 and claudin-1 in culture lysates derived from T. denticola-exposed MDCK cells, suggesting a bacteria-induced protease capable of cleaving these tight junction proteins.

Periodontal Pathogens and Associated Intrathecal Antibodies in Early Stages of Alzheimer's Disease.

BACKGROUND: Recent studies suggest a link between periodontitis and Alzheimer's disease (AD). OBJECTIVE: Verification of the presence of periodontal pathogens and the intrathecal generation of pathogen-specific antibodies in 20 patients with AD and 20 with other forms of dementia (DEM-noAD). METHODS: Clinical periodontal indices were recorded. Cerebrospinal fluid (CSF) was analyzed for total tau protein (T-tau) and amyloid-ß (Aß1-42). In serum and CSF, antibody levels against Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, and Treponema species were quantified. The presence of selected bacteria and inflammatory biomarkers were determined in periodontium, serum, and CSF. RESULTS: In line with diagnoses, CSF-levels of Aß1-42 were significantly lower in AD than DEM-noAD patients. Periodontal destruction and inflammation were omnipresent with no difference between groups. P. gingivalis, T. forsythia, and Treponema species were detected in more than 50% of subgingival biofilm samples, but neither in serum nor in the CSF. Elevated levels of anti-pathogen antibodies in CSF of 16 patients (7 AD; 9 DEM-noAD) compared to serum highlight a possibility of the intrathecal immune response to pathogens. There was no significant difference in antibodies levels against selected bacteria in CSF and serum between groups. Multivariate regression analysis and general linear models revealed an association of the T-tau level in AD group with both serum levels of anti-P. gingivalis antibodies and MCP-1/CCL-2. CONCLUSION: Periodontal pathogens may enter the brain and stimulate a local immune response. However, in patients with dementia at the age up to 70 years, periodontal pathogens do not act as a trigger for developing AD.

Characterization of a novel potential peptide import system in Treponema denticola.

Treponema denticola is a major etiologic agent of chronic periodontitis. On the outer sheath of T. denticola, several proteins, such as the major outer sheath protein and dentilisin were detected, and among them, a 95 kDa protein which has not yet been characterized. The aim of this study was to characterize the function of this 95 kDa protein containing gene cluster. A gene encoding this 95 kDa protein (TDE_1072) of T. denticola was inactivated by homologous recombination. We compared growth curves between the TDE_1072 mutant and wild-type strains as well as differences in gene expression by DNA microarray analysis. Differential expression of genes identified by microarray analysis was confirmed by quantitative reverse transcription-polymerase chain reaction. The proteins encoded by TDE_1072, TDE_1073, TDE_1074, TDE_1075, and TDE_1076 shared respective similarities to the substrate-binding domain (DppA) of an ABC-type dipeptide/oligopeptide/nickel transport system, and to the permease components (DppB and DppC) and ATPase components (DppD and DppF) of an ABC-type dipeptide/oligopeptide/nickel transport system. Inactivation of dppA attenuated the growth of T. denticola and dppA-dppF were co-transcribed. In contrast, expression of oppB-oppF was up-regulated in the mutant. Our findings indicate that TDE_1072 may be a potential periplasmic solute binding protein encoded by dppA that is involved in the organization of a peptide uptake system with dppB-dppF.

Treponema denticola transcriptional profiles in serum-restricted conditions.

Treponema denticola is a major pathogen in periodontal disease and is frequently isolated from the lesions of patients with chronic periodontitis. Treponema denticola utilizes serum components as nutrient sources so as to colonize and proliferate in the gingival crevice. However, the mechanisms of serum utilization remain unclear. Therefore, the aim of the present study was to identify T. denticola serum utilization genes. Precultured T. denticola cells were suspended in a tryptone-yeast extract-gelatin-volatile fatty acids medium containing 0, 1% and 10% serum, respectively, and incubated anaerobically for 17 h. Total RNA was isolated, and T. denticola gene expression was compared by microarray and reverse transcription-polymerase chain reaction. In serum-depleted conditions, the expression levels of a potential hydroxylamine reductase, several ABC transporters, and phosphoenolpyruvate synthase were increased, while those of genes encoding methyl-accepting chemotaxis proteins and a transcriptional regulator were decreased. These results suggest that T. denticola may uptake serum components mainly through the action of ABC transporters. In particular, the decrease in the dmcA expression level with decreasing serum concentration suggests its involvement in chemotaxis toward serum-rich environments.

Plasminogen binding and degradation by Treponema denticola: Identification of the plasminogen binding interface on the FhbB protein.

Treponema denticola is a proteolytic-anaerobic spirochete whose abundance in the subgingival crevice correlates with periodontal disease severity. Treponema denticola evades serum-mediated killing through the binding of factor H (FH), a negative regulator of the complement system. The T. denticolaFH receptor has been identified as FhbB, an 11.4kDa immunodominant lipoprotein. Three distinct subfamilies of FhbB proteins have been delineated and designated as FhbB1, FhbB2 and FhbB3. In this study we demonstrate that all FhbB variants bind human plasminogen (Plg). Competitive binding analyses revealed that FH and Plg do not compete for binding. Binding studies with FhbB135405 site-directed amino acid substitution mutants demonstrated that the interaction domains for FH and Plg on FhbB are separable. Inhibition of Plg-FhbB binding by ε-aminocaproic acid (a lysine analog) indicates that binding is mediated by electrostatic interactions that presumably occur with Lys binding sites contained within Plg "Kringle" domains 1, 2, 4 or 5. Similar to that demonstrated for FH, Plg can also serve as a substrate for the T. denticola protease, dentilisin. The in vivo consequences of dentilisin-mediated cleavage of Plg remained to be determined. The data presented demonstrate that FhbB is a multi-functional protein that may contribute to virulence through several mechanisms including immune evasion, manipulation of the host immune response, adherence or tissue invasion.

Caspase-4 activation by a bacterial surface protein is mediated by cathepsin G in human gingival fibroblasts.

Caspase-4 is an inflammatory caspase; however, its mechanism of activation is poorly understood. In this study, we demonstrate that Td92, a surface protein of the periodontal pathogen Treponema denticola and a homolog of the Treponema pallidum surface protein Tp92, activates caspase-4 and induces pyroptosis in primary cultured human gingival fibroblasts (HGFs) via cathepsin G activation. Cathepsin G inhibition or siRNA knockdown of cathepsin G inhibited Td92-induced caspase-4 activation and cell death. Td92-induced cell death was significantly inhibited by siRNA knockdown of gasdermin D. Td92 treatment resulted in the binding of cathepsin G to caspase-4 and the coaggregation of these two molecules. In addition, Td92 induced IL-1α expression and secretion, and this was inhibited by caspase-4 knockdown. Cytochalasin D did not block Td92-induced caspase-4 activation, suggesting that Td92 internalization is not required for caspase-4 activation. Our results demonstrate that cathepsin G is directly engaged in caspase-4 activation by a bacterial ligand, which is responsible for cell death and IL-1α secretion in HGFs.

The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola.

The major outer sheath protein (MOSP) is a prominent constituent of the cell envelope of Treponema denticola (TDE) and one of its principal virulence determinants. Bioinformatics predicts that MOSP consists of N- and C-terminal domains, MOSPN and MOSPC. Biophysical analysis of constructs refolded in vitro demonstrated that MOSPC, previously shown to possess porin activity, forms amphiphilic trimers, while MOSPN forms an extended hydrophilic monomer. In TDE and E. coli expressing MOSP with a PelB signal sequence (PelB-MOSP), MOSPC is OM-embedded and surface-exposed, while MOSPN resides in the periplasm. Immunofluorescence assay, surface proteolysis, and novel cell fractionation schemes revealed that MOSP in TDE exists as outer membrane (OM) and periplasmic trimeric conformers; PelB-MOSP, in contrast, formed only OM-MOSP trimers. Although both conformers form hetero-oligomeric complexes in TDE, only OM-MOSP associates with dentilisin. Mass spectrometry (MS) indicated that OM-MOSP interacts with proteins in addition to dentilisin, most notably, oligopeptide-binding proteins (OBPs) and the ß-barrel of BamA. MS also identified candidate partners for periplasmic MOSP, including TDE1658, a spirochete-specific SurA/PrsA ortholog. Collectively, our data suggest that MOSP destined for the TDE OM follows the canonical BAM pathway, while formation of a stable periplasmic conformer involves an export-related, folding pathway not present in E. coli.

Infection and apoptosis associated with inflammation in periodontitis: An immunohistologic study.

OBJECTIVE: Evidence of increased apoptosis is observed in periodontitis and may be associated with destruction of the periodontal tissue caused by the increased cell death, with the release of danger signals and subsequent stimulation of the proinflammatory processes. However, the exact mechanisms associated with these processes remain unclear. This study aimed to investigate the presence of the periodontal pathogen Treponema denticola, apoptosis, high mobility group box 1 as a damage-associated molecular pattern, and several inflammatory markers in periodontitis and gingivitis subjects. MATERIALS AND METHODS: Soft tissue specimens from gingival tissues of periodontitis and gingivitis patients were used for immunohistochemical and immunofluorescence staining of T. denticola chymotrypsin-like proteinase (CTLP), apoptosis markers, high mobility group box 1, Toll-like receptor 4, inflammatory cell markers, and proinflammatory cytokines. RESULTS: Treponema denticola was detected in all periodontitis-affected tissues. This was associated with a significant increase in the number of apoptotic cells, including macrophages, alterations in the expression of high mobility group box 1 and its receptor, and increased levels of proinflammatory cytokines compared with gingivitis. CONCLUSIONS: In summary, the presence of T. denticola (especially its CTLP), apoptosis, high mobility group box 1, and inflammatory markers suggests their potential involvement in the pathogenesis of periodontitis.

The C-terminal region of the major outer sheath protein of Treponema denticola inhibits neutrophil chemotaxis.

Treponema denticola is an oral spirochete strongly associated with severe periodontal disease. A prominent virulence factor, the major outer sheath protein (Msp), disorients neutrophil chemotaxis by altering the cellular phosphoinositide balance, leading to impairment of downstream chemotactic events including actin rearrangement, Rac1 activation, and Akt activation in response to chemoattractant stimulation. The specific regions of Msp responsible for interactions with neutrophils remain unknown. In this study, we investigated the inhibitory effect of truncated Msp regions on neutrophil chemotaxis and associated signaling pathways. Murine neutrophils were treated with recombinant protein truncations followed by assessment of chemotaxis and associated signal pathway activation. Chemotaxis assays indicate sequences within the C-terminal region; particularly the first 130 amino acids, have the strongest inhibitory effect on neutrophil chemotaxis. Neutrophils incubated with the C-terminal region protein also demonstrated the greatest inhibition of Rac1 activation, increased phosphoinositide phosphatase activity, and decreased Akt activation; orchestrating impairment of chemotaxis. Furthermore, incubation with antibodies specific to only the C-terminal region blocked the Msp-induced inhibition of chemotaxis and denaturing the protein restored Rac1 activation. Msp from the strain OTK, with numerous amino acid substitutions throughout the polypeptide, including the C-terminal region compared with strain 35405, showed increased ability to impair neutrophil chemotaxis. Collectively, these results indicate that the C-terminal region of Msp is the most potent region to modulate neutrophil chemotactic signaling and that specific sequences and structures are likely to be required. Knowledge of how spirochetes dampen the neutrophil response is limited and Msp may represent a novel therapeutic target for periodontal disease.

A novel glycan modifies the flagellar filament proteins of the oral bacterium Treponema denticola.

While protein glycosylation has been reported in several spirochetes including the syphilis bacterium Treponema pallidum and Lyme disease pathogen Borrelia burgdorferi, the pertinent glycan structures and their roles remain uncharacterized. Herein, a novel glycan with an unusual chemical composition and structure in the oral spirochete Treponema denticola, a keystone pathogen of periodontitis was reported. The identified glycan of mass 450.2 Da is composed of a monoacetylated nonulosonic acid (Non) with a novel extended N7 acyl modification, a 2-methoxy-4,5,6-trihydroxy-hexanoyl residue in which the Non has a pseudaminic acid configuration (L-glycero-L-manno) and is ß-linked to serine or threonine residues. This novel glycan modifies the flagellin proteins (FlaBs) of T. denticola by O-linkage at multiple sites near the D1 domain, a highly conserved region of bacterial flagellins that interact with Toll-like receptor 5. Furthermore, mutagenesis studies demonstrate that the glycosylation plays an essential role in the flagellar assembly and motility of T. denticola. To our knowledge, this novel glycan and its unique modification sites have not been reported previously in any bacteria.

1,2-Diacylglycerol choline phosphotransferase catalyzes the final step in the unique Treponema denticola phosphatidylcholine biosynthesis pathway.

Treponema denticola synthesizes phosphatidylcholine through a licCA-dependent CDP-choline pathway identified only in the genus Treponema. However, the mechanism of conversion of CDP-choline to phosphatidylcholine remained unclear. We report here characterization of TDE0021 (herein designated cpt) encoding a 1,2-diacylglycerol choline phosphotransferase homologous to choline phosphotransferases that catalyze the final step of the highly conserved Kennedy pathway for phosphatidylcholine synthesis in eukaryotes. T. denticola Cpt catalyzed in vitro phosphatidylcholine formation from CDP-choline and diacylglycerol, and full activity required divalent manganese. Allelic replacement mutagenesis of cpt in T. denticola resulted in abrogation of phosphatidylcholine synthesis. T. denticola Cpt complemented a Saccharomyces cerevisiae CPT1 mutant, and expression of the entire T. denticola LicCA-Cpt pathway in E. coli resulted in phosphatidylcholine biosynthesis. Our findings show that T. denticola possesses a unique phosphatidylcholine synthesis pathway combining conserved prokaryotic choline kinase and CTP:phosphocholine cytidylyltransferase activities with a 1,2-diacylglycerol choline phosphotransferase that is common in eukaryotes. Other than in a subset of mammalian host-associated Treponema that includes T. pallidum, this pathway is found in neither bacteria nor Archaea. Molecular dating analysis of the Cpt gene family suggests that a horizontal gene transfer event introduced this gene into an ancestral Treponema well after its divergence from other spirochetes.

Spirochaete flagella hook proteins self-catalyse a lysinoalanine covalent crosslink for motility.

Spirochaetes are bacteria responsible for several serious diseases, including Lyme disease (Borrelia burgdorferi), syphilis (Treponema pallidum) and leptospirosis (Leptospira interrogans), and contribute to periodontal diseases (Treponema denticola)(1). These spirochaetes employ an unusual form of flagella-based motility necessary for pathogenicity; indeed, spirochaete flagella (periplasmic flagella) reside and rotate within the periplasmic space(2-11). The universal joint or hook that links the rotary motor to the filament is composed of ∼120-130 FlgE proteins, which in spirochaetes form an unusually stable, high-molecular-weight complex(9,12-17). In other bacteria, the hook can be readily dissociated by treatments such as heat(18). In contrast, spirochaete hooks are resistant to these treatments, and several lines of evidence indicate that the high-molecular-weight complex is the consequence of covalent crosslinking(12,13,17). Here, we show that T. denticola FlgE self-catalyses an interpeptide crosslinking reaction between conserved lysine and cysteine, resulting in the formation of an unusual lysinoalanine adduct that polymerizes the hook subunits. Lysinoalanine crosslinks are not needed for flagellar assembly, but they are required for cell motility and hence infection. The self-catalytic nature of FlgE crosslinking has important implications for protein engineering, and its sensitivity to chemical inhibitors provides a new avenue for the development of antimicrobials targeting spirochaetes.

Interaction of spirochetes with the host fibrinolytic system and potential roles in pathogenesis.

The pathogenic spirochetes Borrelia burgdorferi, B. hermsii, B. recurrentis, Treponema denticola and Leptospira spp. are the etiologic agents of Lyme disease, relapsing fever, periodontitis and leptospirosis, respectively. Lyme borreliosis is a multi-systemic disorder and the most prevalent tick-borne disease in the northern hemisphere. Tick-borne relapsing fever is persistent in endemic areas worldwide, representing a significant burden in some African regions. Periodontal disease, a chronic inflammatory disorder that often leads to tooth loss, is caused by several potential pathogens found in the oral cavity including T. denticola. Leptospirosis is considered the most widespread zoonosis, and the predominant human disease in tropical, undeveloped regions. What these diseases have in common is that they are a significant burden to healthcare costs in the absence of prophylactic measures. This review addresses the interaction of these spirochetes with the fibrinolytic system, plasminogen (Plg) binding to the surface of bacteria and the generation of plasmin (Pla) on their surface. The consequences on host-pathogen interactions when the spirochetes are endowed with this proteolytic activity are discussed on the basis of the results reported in the literature. Spirochetes equipped with Pla activity have been shown to degrade extracellular matrix (ECM) components, in addition to digesting fibrin, facilitating bacterial invasion and dissemination. Pla generation triggers the induction of matrix metalloproteases (MMPs) in a cascade of events that enhances the proteolytic capacity of the spirochetes. These activities in concert with the interference exerted by the Plg/Pla on the complement system - helping the bacteria to evade the immune system - should illuminate our understanding of the mechanisms involved in host infection.

Fibronectin-binding protein TDE1579 affects cytotoxicity of Treponema denticola.

While FbpA, a family of bacterial fibronectin (FN) binding proteins has been studied in several gram-positive bacteria, the gram-negative Treponema denticola, an anaerobic periodontal pathogen, also has an overlooked fbp gene (tde1579). In this research, we confirm that recombinant Fbp protein (rFbp) of T. denticola binds human FN with a Kdapp of 1.5 × 10(-7) M and blocks the binding of T. denticola to FN in a concentration-dependent manner to a level of 42%. The fbp gene was expressed in T. denticola. To reveal the roles of fbp in T. denticola pathogenesis, an fbp isogenic mutant was constructed. The fbp mutant had 51% reduced binding ability to human gingival fibroblasts (hGF). When hGF were challenged with T. denticola, the fbp mutant caused less cell morphology change, had 50% reduced cytotoxicity to hGF, and had less influence on the growth of hGF cells.