Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using ¹³C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.     

Antigenic Behaviors of Two Axial Flagellar Proteins Detected in Treponema denticola

Two polypeptide antigens with molecular sizes of 34,000 daltons (34 kDa) and 38 kDa were separated from heated cells of a human clinical treponeme strain G7201 and Treponema denticola ATCC 35404, respectively. The rabbit polyclonal antisera against these antigens were produced and examined for their immunological reactions with the two heated antigens or intact spirochetal cells. Immunoblot analysis showed that the 34-kDa protein was also detected in T. denticola ATCC 35404 and ATCC 33520, and the 38-kDa protein was detected only in the two ATCC strains. Immunoelectron microscopy using the two rabbit antisera and protein A-gold complexes demonstrated that the 38-kDa protein antigen was present on the axial flagella of two T. denticola strains, and that the 34-kDa protein was located in the axial flagella of the G7201 cell, but neither in axial flagella nor on outer envelopes of the two ATCC strains cells, suggesting that the native 34-kDa axial flagellar protein of the G7201 strain may be different from that of T. denticola in terms of immunological reactivity.     

Development of a Transposon Mutagenesis System in the Oral Spirochete Treponema denticola

Here, we report successful transposon mutagenesis in the oral spirochete Treponema denticola. A modified Himar1 transposon, including a new antibiotic selection cassette for T. denticola, generated mutations affecting cell division, transport, and chemotaxis, among other processes. This random mutagenesis system should facilitate research on the biology and pathogenesis of this spirochete, which is associated with human periodontal diseases.     

Discovery of Bovine Digital Dermatitis-Associated Treponema spp. in the Dairy Herd Environment by a Targeted Deep-Sequencing Approach

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.     

Cleavage of Human Immunoglobulin G by Treponema denticola

Mechanisms by which microbial proteases may counteract the local host immune system include the degradation of immunoglobulins. In this study, we report the capacity of the periodontopathogen Treponema denticola to degrade immunoglobulin G (IgG). Intact IgG was not hydrolysed by whole cells, as revealed by SDS-PAGE analysis. When IgG molecules were treated with endoglycosidase F to remove the carbohydrate moiety, significant degradation was observed. However, pre-treatment with glycosidases possessing specificities different from endoglycosidase F (lysozyme or neuraminidase) did not render the molecule susceptible to cleavage by T. denticola. SDS-PAGE analysis of the IgG degradation products suggests that T. denticola cleaves inside the heavy chain polypeptide. Serine-specific protease inhibitors were highly effective in inhibiting the degradation of glycosidase-treated IgG molecules by T. denticola. The synergistic effect of glycolytic enzymes andT. denticola proteases on IgG may occur during periodontitis since both glycolytic activities and spirochete numbers significantly increase in diseased periodontal sites.     

Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.     

PCR analysis of Actinobacillus actinomycetemcomitans,Porphyromonas gingivalis,Treponema denticola andFusobacterium nucleatum in middle ear effusion

Anaerobes contribute to the severity and chronicity of infections that occur in and around the oral cavity. One of the factors involved in the pathogenesis of otitis media with effusion (OME) is the retrograde movement of bacteria from the oropharynx into the middle ear cavity. OME is one of the most common causes of hearing loss in children. We have used a PCR-based method to identify Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola andFusobacterium nucleatum in 65 middle ear effusion (MEE) samples obtained from paediatric patients seen for myringotomy and tube placement. DNA was extracted from MEE samples and PCR was initially done with DNA extracts by using the universal primers within the 16S rRNA gene sequence common to all bacterial species. The positive samples were further assessed with four species-specific primers. With the universal primers, 27 of 65 samples (41.5%) showed positive reaction indicating the presence of bacterial DNA. F. nucleatum was present in 10 out of 27 PCR-positive samples (37%) while one sample was positive for both T. denticola and F. nucleatum (3.7%). A. actinomycetemcomitans and P. gingivalis were not detected in any of the samples. The results of this study suggest that oral bacterial species may also play a role in the aetiopathogenesis of paediatric MEE.     

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lacto-ferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29–34 kDa protein.     

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.     

A surface‐exposed neuraminidase affects complement resistance and virulence of the oral spirochaete Treponema denticola

Neuraminidases (sialidases) catalyse the removal of terminal sialic acid from glycoconjugates. Bacterial pathogens often utilize neuraminidases to scavenge host sialic acid, which can be utilized either as a nutrient or as a decorating molecule to disguise themselves from host immune attacks. Herein, a putative neuraminidase (TDE0471) was identified in Treponema denticola, an oral spirochaete associated with human periodontitis. TDE0471 is a cell surface‐exposed exo‐neuraminidase that removes sialic acid from human serum proteins; it is required for T. denticola to grow in a medium that mimics gingival crevice fluid, suggesting that the spirochaete may use sialic acid as a nutrient in vivo. TDE0471 protects T. denticola from serum killing by preventing the deposition of membrane attack complexes on the bacterial cell surface. Animal studies revealed that a TDE0471‐deficient mutant is less virulent than its parental wild‐type strain in BALB/C mice. However, it causes a level of tissue damage similar to the wild type in complement‐deficient B6.129S4‐C3^tm1Crr/J mice albeit the damage caused by both bacterial strains is more severe in these transgenic mice. Based on these results, we propose that T. denticola has evolved a strategy to scavenge host sialic acid using its neuraminidase, which allows the spirochaete to acquire nutrients and evade complement killing.     

Evidence that TP_0144 of Treponema pallidum Is a Thiamine-Binding Protein

Thiamine pyrophosphate (TPP), the biologically active form of thiamine (also known as vitamin B₁), is an essential cofactor for several important enzymes involved in carbohydrate metabolism, and therefore, it is required for all living organisms. We recently found that a thiamine-binding protein (TDE_0143) is essential for the survival of Treponema denticola, an important bacterial pathogen that is associated with human periodontitis. In this report, we provide experimental evidence showing that TP_0144, a homolog of TDE_0143 from the syphilis spirochete Treponema pallidum, is a thiamine-binding protein that has biochemical features and functions that are similar to those of TDE_0143. First, structural modeling analysis reveal that both TDE_0143 and TP_0144 contain a conserved TPP-binding site and share similar structures to the thiamine-binding protein of Escherichia coli. Second, biochemical analysis shows that these two proteins bind to TPP with similar dissociation constant (K_d) values (TDE_0143, K_d of 36.50 nM; TP_0144, K_d of 32.62 nM). Finally, heterologous expression of TP_0144 in a ΔTDE_0143 strain, a previously constructed TDE_0143 mutant of T. denticola, fully restores its growth and TPP uptake when exogenous thiamine is limited. Collectively, these results indicate that TP_0144 is a thiamine-binding protein that is indispensable for T. pallidum to acquire exogenous thiamine, a key nutrient for bacterial survival. In addition, the studies shown in this report further underscore the feasibility of using T. denticola as a platform to study the biology and pathogenicity of T. pallidum and probably other uncultivable treponemal species as well.     

Comparison of peptidase, glycosidase and esterase activities of oral and non-oral Treponema species

The enzyme profiles of 20 oral and non-oral Treponema strains were investigated using an API ZYM Complete Research kit. The test included 10 2-naphthyl derivatives of fatty acids, 20 p-nitrophenol derivatives of carbohydrates and 60 2-naphthylamide derivatives of amino acids and peptides. The oral Treponema species investigated were T. denticola, T. vincentii and T. Pectinovorum. The non-oral species examined were T. phagedenis, T. hyodysenteriae and intestinal spirochaetes of human and chicken origin. Esterase activities on C5 to C10 fatty acids were common among different Treponema species. Glycosidase activities were infrequently observed in T. vincentii, T. pectinovorum and T. phagedenis Reiter strain. Arabinosidase, lactosidase and xylosidase activity was observed in the T. hyodysenteriae strains but alpha-L-fucosidase activity was found only in T. denticola and T. phagedenis. More exo- and endo-peptidase activities were found in T. denticola than in other species. The enteropathogenic T. hyodysenteriae isolates had a very low proteolytic profile. Dipeptidyl prolyl amidase activity was observed in all species except in the T. phagedenis Reiter strain and the avian intestinal spirochaetes. The enzyme profiles did not discriminate between oral and non-oral Treponema species.     

Fibronectin-Binding Proteins of a Human Oral Spirochete Treponema denticola

Major polypeptides from a human oral spirochete Treponema denticola ATCC 33520 were examined to demonstrate their ability to bind to human plasma fibronectin by immunoblot analysis. Of three main polypeptides separated on sodium dodecyl sulfate polyacrylamide gels 53,000-daltons (53-kDa) and 72-kDa surface antigenic proteins and a 38-kDa axial flagellar protein showed the ability to bind to fibronectin, suggesting that fibronectin on host cells can mediate cytoadherence of T. denticola by its binding to the surface proteins or the exposed 38-kDa axial flageller protein.     

The Gastrointestinal Tract as a Potential Infection Reservoir of Digital Dermatitis-Associated Treponemes in Beef Cattle and Sheep

Digital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium-like, Treponema phagedenis-like, and Treponema pedis. The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival (n = 40) and rectal (n = 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes (T. pedis), as were 3/40 rectal tissues (one containing T. medium-like and two containing T. pedis). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues (n = 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis-like). A T. phagedenis-like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle (n = 41) and sheep (n = 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species.     

pyrF as a Counterselectable Marker for Unmarked Genetic Manipulations in Treponema denticola

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5′-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgEⁱⁿ) regained the susceptibility to 5-FOA. Finally, FlgEⁱⁿ was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE^out) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE^out was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE^out has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.     

Dentilisin Activity Affects the Organization of the Outer Sheath of Treponema denticola

Prolyl-phenylalanine-specific serine protease (dentilisin) is a major extracellular protease produced by Treponema denticola. The gene, prtP, coding for the protease was recently cloned and sequenced (K. Ishihara, T. Miura, H. K. Kuramitsu, and K. Okuda, Infect. Immun. 64:5178–5186, 1996). In order to determine the role of this protease in the physiology and virulence of T. denticola, a dentilisin-deficient mutant, K1, was constructed following electroporation with a prtP-inactivated DNA fragment. No chymotrypsin-like protease activity was detected in the dentilisin-deficient mutant. In addition, the high-molecular-mass oligomeric protein characteristic of the outer sheath of the organism decreased in the mutant. Furthermore, the hydrophobicity of the mutant was decreased, and coaggregation of the mutant with Fusobacterium nucleatum was enhanced compared to that of the wild-type organism. The results obtained with a mouse abscess model system indicated that the virulence of the mutant was attenuated relative to that of the wild-type organism. These results suggest that dentilisin activity plays a major role in the structural organization of the outer sheath of T. denticola. The loss of dentilsin activity and the structural change in the outer sheath affect the pathogenicity of T. denticola.     

A simplified erythromycin resistance cassette for Treponema denticola mutagenesis

The primary selectable marker for the genetic studies of Treponema denticola is a hybrid gene cassette containing both ermF and ermAM (ermB) genes. ErmB functions in Escherichia coli, while ErmF has been assumed to confer resistance in T. denticola. We demonstrate here that ErmB is sufficient for erythromycin selection in T. denticola and that the native ermB promoter drives ErmB expression.     

Adherence of Human Oral Spirochetes by Collagen-Binding Proteins

The ability of spirochetes to adhere to collagens was compared among three species of human oral treponemes. Immunoblot analysis demonstrated that type I-, IV-, and V-collagen-binding polypeptides (CBPs) were detected in the heated and unheated preparations from both Treponema denticola ATCC 33520 and T. socranskii subsp. buccale ATCC 35534. Few CBPs, however, were detected in the heated and unheated preparations from a recently characterized isolate, T. medium strain G7201. Immunoelectron microscopy using rabbit antisera against the CBPs from the unheated preparations demonstrated that four CBPs, a 27 kDa type V-CBP of T. denticola ATCC 33520, a 95 kDa type IV-CBP and a 110 kDa type I-CBP of T. socranskii subsp. buccale ATCC 35534, and a 95 kDa type IV-CBP of T. medium strain G7201, were located on the outer envelopes of the individual cells. The adherence of T. denticola to the collagen-coated surfaces was significantly greater than that of T. medium, suggesting that the CBPs on the oral spirochetal cells play an important role in their adherence to collagen-rich connective tissues of the host.     

Altered Microbiomes in Bovine Digital Dermatitis Lesions,and the Gut as a Pathogen Reservoir

Bovine digital dermatitis (DD) is the most important infectious disease associated with lameness in cattle worldwide. Since the disease was first described in 1974, a series of Treponema species concurrent with other microbes have been identified in DD lesions, suggesting a polymicrobial etiology. However, the pathogenesis of DD and the source of the causative microbes remain unclear. Here we characterized the microbiomes of healthy skin and skin lesions in dairy cows affected with different stages of DD and investigated the gut microbiome as a potential reservoir for microbes associated with this disease. Discriminant analysis revealed that the microbiomes of healthy skin, active DD lesions (ulcerative and chronic ulcerative) and inactive DD lesions (healing and chronic proliferative) are completely distinct. Treponema denticola, Treponema maltophilum, Treponema medium, Treponema putidum, Treponema phagedenis and Treponema paraluiscuniculi were all found to be present in greater relative abundance in active DD lesions when compared with healthy skin and inactive DD lesions, and these same Treponema species were nearly ubiquitously present in rumen and fecal microbiomes. The relative abundance of Candidatus Amoebophilus asiaticus, a bacterium not previously reported in DD lesions, was increased in both active and inactive lesions when compared with healthy skin. In conclusion, our data support the concept that DD is a polymicrobial disease, with active DD lesions having a markedly distinct microbiome dominated by T. denticola, T. maltophilum, T. medium, T. putidum, T. phagedenis and T. paraluiscuniculi. Furthermore, these Treponema species are nearly ubiquitously found in rumen and fecal microbiomes, suggesting that the gut is an important reservoir of microbes involved in DD pathogenesis. Additionally, the bacterium Candidatus Amoebophilus asiaticus was highly abundant in active and inactive DD lesions.