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Treponema denticola, a keystone pathogen in periodontitis, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp forms an oligomeric outer membrane complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular processes in host cells. T. denticola msp is an ortholog of the Treponema pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. We recently identified the primary Msp surface-exposed epitope and proposed a model of the Msp protein as a β-barrel protein similar to Gram-negative bacterial porins. Here, we report fine-scale Msp mutagenesis demonstrating that both the N and C termini as well as the centrally located Msp surface epitope are required for native Msp oligomer expression. Removal of as few as three C-terminal amino acids abrogated Msp detection on the T. denticola cell surface, and deletion of four residues resulted in complete loss of detectable Msp. Substitution of a FLAG tag for either residues 6 to 13 of mature Msp or an 8-residue portion of the central Msp surface epitope resulted in expression of full-length Msp but absence of the oligomer, suggesting roles for both domains in oligomer formation. Consistent with previously reported Msp N-glycosylation, proteinase K treatment of intact cells released a 25 kDa polypeptide containing the Msp surface epitope into culture supernatants. Molecular modeling of Msp using novel metagenome-derived multiple sequence alignment (MSA) algorithms supports the hypothesis that Msp is a large-diameter, trimeric outer membrane porin-like protein whose potential transport substrate remains to be identified. IMPORTANCE The Treponema denticola gene encoding its major surface protein (Msp) is an ortholog of the T. pallidum tprA to -K gene family that includes tprK, whose remarkable in vivo hypervariability is proposed to contribute to T. pallidum immune evasion. Using a combined strategy of fine-scale mutagenesis and advanced predictive molecular modeling, we characterized the Msp protein and present a high-confidence model of its structure as an oligomer embedded in the outer membrane. This work adds to knowledge of Msp-like proteins in oral treponemes and may contribute to understanding the evolutionary and potential functional relationships between T. denticola Msp and the orthologous T. pallidum Tpr proteins.

The oral spirochete Treponema denticola is a keystone periodontal pathogen that, in association with members of a complex polymicrobial oral biofilm, contributes to tissue damage and alveolar bone loss in periodontal disease. Virulence-associated behaviors attributed to T. denticola include disruption of the host cell extracellular matrix, tissue penetration and disruption of host cell membranes accompanied by dysregulation of host immunoregulatory factors. T. denticola dentilisin is associated with several of these behaviors. Dentilisin is an outer membrane-associated complex of acylated subtilisin-family PrtP protease and two other lipoproteins, PrcB and PrcA, that are unique to oral spirochetes. Dentilisin is encoded in a single operon consisting of prcB-prcA-prtP. We employ multiple approaches to study mechanisms of dentilisin assembly and PrtP protease activity. To determine the role of each protein in the protease complex, we have made targeted mutations throughout the protease locus, including polar and nonpolar mutations in each gene (prcB, prcA, prtP) and deletions of specific PrtP domains, including single base mutagenesis of key PrtP residues. These will facilitate distinguishing between host cell responses to dentilisin protease activity and its acyl groups. The boundaries of the divergent promoter region and the relationship between dentilisin and the adjacent iron transport operon are being resolved by incremental deletions in the sequence immediately 5’ to the protease locus. Comparison of the predicted three-dimensional structure of PrtP to that of other subtilisin-like proteases shows a unique PrtP C-terminal domain of approximately 250 residues. A survey of global gene expression in the presence or absence of protease gene expression reveals potential links between dentilisin and iron uptake and homeostasis in T. denticola. Understanding the mechanisms of dentilisin transport, assembly and activity of this unique protease complex may lead to more effective prophylactic or therapeutic treatments for periodontal disease.

The availability of divalent metal cations required as cofactors for microbial metabolism is severely limited in the host environment. Bacteria have evolved highly regulated uptake systems to maintain essential metal homeostasis to meet cellular demands while preventing toxicity. The Tro operon (troABCDR), present in all sequenced Treponema spp., is a member of a highly conserved family of ATP-binding cassette transporters involved in metal cation uptake whose expression is controlled by TroR, a DtxR-like cation-responsive regulatory protein. Transcription of troA responds to divalent manganese and iron (T. denticola) or manganese and zinc (T. pallidum), and metal-dependent TroR binding to the troA promoter represses troA transcription. We report here the construction and complementation of defined T. denticola ΔtroR and ΔtroA strains to characterize (i) the role of TroA in metal-dependent T. denticola growth and (ii) the role of TroR in T. denticola gene expression. We show that TroA expression is required for T. denticola growth under iron- and manganese-limited conditions. Furthermore, TroR is required for the transcriptional regulation of troA in response to iron or manganese, and deletion of troR results in significant differential expression of more than 800 T. denticola genes in addition to troA. These results suggest that (i) TroA-mediated cation uptake is important in metal homeostasis in vitro and may be important for Treponema survival in the host environment and (ii) the absence of TroR results in significant dysregulation of nearly one-third of the T. denticola genome. These effects may be direct (as with troA) or indirect due to dysregulation of metal homeostasis. IMPORTANCETreponema denticola is one of numerous host-associated spirochetes, a group including commensals, pathobionts, and at least one frank pathogen. While most T. denticola research concerns its role in periodontitis, its relative tractability for growth and genetic manipulation make it a useful model for studying Treponema physiology, metabolism, and host-microbe interactions. Metal micronutrient acquisition and homeostasis are highly regulated both in microbial cells and by host innate defense mechanisms that severely limit metal cation bioavailability. Here, we characterized the T. denticolatroABCDR operon, the role of TroA-mediated iron and manganese uptake in growth, and the effects of TroR on global gene expression. This study contributes to our understanding of the mechanisms involved in cellular metal homeostasis required for survival in the host environment.

The availability of divalent metal cations required as cofactors for microbial metabolism is severely limited in the host environment. Bacteria have evolved highly regulated uptake systems to maintain essential metal homeostasis to meet cellular demands while preventing toxicity. The Tro operon (troABCDR), present in all sequenced Treponema spp., is a member of a highly conserved family of ATP-binding cassette transporters involved in metal cation uptake whose expression is controlled by TroR, a DtxR-like cation-responsive regulatory protein. Transcription of troA responds to divalent manganese and iron (T. denticola) or manganese and zinc (T. pallidum), and metal-dependent TroR binding to the troA promoter represses troA transcription. We report here the construction and complementation of defined T. denticola ΔtroR and ΔtroA strains to characterize (i) the role of TroA in metal-dependent T. denticola growth and (ii) the role of TroR in T. denticola gene expression. We show that TroA expression is required for T. denticola growth under iron- and manganese-limited conditions. Furthermore, TroR is required for the transcriptional regulation of troA in response to iron or manganese, and deletion of troR results in significant differential expression of more than 800 T. denticola genes in addition to troA. These results suggest that (i) TroA-mediated cation uptake is important in metal homeostasis in vitro and may be important for Treponema survival in the host environment and (ii) the absence of TroR results in significant dysregulation of nearly one-third of the T. denticola genome. These effects may be direct (as with troA) or indirect due to dysregulation of metal homeostasis. IMPORTANCE Treponema denticola is one of numerous host-associated spirochetes, a group including commensals, pathobionts, and at least one frank pathogen. While most T. denticola research concerns its role in periodontitis, its relative tractability for growth and genetic manipulation make it a useful model for studying Treponema physiology, metabolism, and host-microbe interactions. Metal micronutrient acquisition and homeostasis are highly regulated both in microbial cells and by host innate defense mechanisms that severely limit metal cation bioavailability. Here, we characterized the T. denticola troABCDR operon, the role of TroA-mediated iron and manganese uptake in growth, and the effects of TroR on global gene expression. This study contributes to our understanding of the mechanisms involved in cellular metal homeostasis required for survival in the host environment.

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.

Treponema denticola, one of several recognized periodontal pathogens, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp (or MOSP) comprises an oligomeric outer membrane-associated complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular responses. There are two hypotheses regarding native Msp structure and membrane topology. One hypothesis predicts that the entire Msp protein forms a β-barrel structure similar to that of well-studied outer membrane porins of Gram-negative bacteria. The second hypothesis predicts a bipartite Msp with distinct and separate periplasmic N-terminal and porin-like β-barrel C-terminal domains. The bipartite model, based on bioinformatic analysis of the orthologous Treponema pallidum Tpr proteins, is supported largely by studies of recombinant TprC and Msp polypeptides. The present study reports immunological studies in both T. denticola and Escherichia coli backgrounds to identify a prominent Msp surface epitope (residues 229 to 251 in ATCC 35405) in a domain that differs between strains with otherwise highly conserved Msps. These results were then used to evaluate a series of in silico structural models of representative T. denticola Msps. The data presented here are consistent with a model of Msp as a large-diameter β-barrel porin. This work adds to the knowledge regarding the diverse Msp-like proteins in oral treponemes and may contribute to an understanding of the evolutionary and potential functional relationships between Msps of oral Treponema and the orthologous group of Tpr proteins of T. pallidum. IMPORTANCETreponema denticola is among a small subset of the oral microbiota contributing to severe periodontal disease. Due to its relative genetic tractability, T. denticola is a model organism for studying Treponema physiology and host-microbe interactions. T. denticola Msp is a highly expressed outer membrane-associated oligomeric protein that binds fibronectin, has cytotoxic pore-forming activity, and disrupts intracellular regulatory pathways. It shares homology with the orthologous group of T. pallidum Tpr proteins, one of which is implicated in T. pallidum in vivo antigenic variation. The outer membrane topologies of both Msp and the Tpr family proteins are unresolved, with conflicting reports on protein domain localization and function. In this study, we combined empirical immunological data derived both from diverse T. denticola strains and from recombinant Msp expression in E. coli with in silico predictive structural modeling of T. denticola Msp membrane topology, to move toward resolution of this important issue in Treponema biology.

Treponema denticola, one of several recognized periodontal pathogens, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp (or MOSP) comprises an oligomeric outer membrane-associated complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular responses. There are two hypotheses regarding native Msp structure and membrane topology. One hypothesis predicts that the entire Msp protein forms a β-barrel structure similar to that of well-studied outer membrane porins of Gram-negative bacteria. The second hypothesis predicts a bipartite Msp with distinct and separate periplasmic N-terminal and porin-like β-barrel C-terminal domains. The bipartite model, based on bioinformatic analysis of the orthologous Treponema pallidum Tpr proteins, is supported largely by studies of recombinant TprC and Msp polypeptides. The present study reports immunological studies in both T. denticola and Escherichia coli backgrounds to identify a prominent Msp surface epitope (residues 229 to 251 in ATCC 35405) in a domain that differs between strains with otherwise highly conserved Msps. These results were then used to evaluate a series of in silico structural models of representative T. denticola Msps. The data presented here are consistent with a model of Msp as a large-diameter β-barrel porin. This work adds to the knowledge regarding the diverse Msp-like proteins in oral treponemes and may contribute to an understanding of the evolutionary and potential functional relationships between Msps of oral Treponema and the orthologous group of Tpr proteins of T. pallidum. IMPORTANCE Treponema denticola is among a small subset of the oral microbiota contributing to severe periodontal disease. Due to its relative genetic tractability, T. denticola is a model organism for studying Treponema physiology and host-microbe interactions. T. denticola Msp is a highly expressed outer membrane-associated oligomeric protein that binds fibronectin, has cytotoxic pore-forming activity, and disrupts intracellular regulatory pathways. It shares homology with the orthologous group of T. pallidum Tpr proteins, one of which is implicated in T. pallidum in vivo antigenic variation. The outer membrane topologies of both Msp and the Tpr family proteins are unresolved, with conflicting reports on protein domain localization and function. In this study, we combined empirical immunological data derived both from diverse T. denticola strains and from recombinant Msp expression in E. coli with in silico predictive structural modeling of T. denticola Msp membrane topology, to move toward resolution of this important issue in Treponema biology.

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.

In mammals, males undergo a greater number of germline cell divisions compared with females. Thus, the male germline accumulates more DNA replication errors, which result in male mutation bias-a higher mutation rate for males than for females. The phenomenon of male mutation bias has been investigated mostly for rodents and primates, however, it has not been studied in detail for other mammalian orders. Here we sequenced and analyzed five introns of three genes (DBX/DBY, UTX/UTY, and ZFX/ZFY) homologous between X and Y chromosomes in several species of perissodactyls (horses and rhinos) and of primates. Male mutation bias was evident: substitution rate was higher for a Y chromosome intron than for its X chromosome homologue for all five intron pairs studied. Substitution rates varied regionally among introns sequenced on the same chromosome and this variation influenced male mutation bias inferred from each intron pair. Interestingly, we observed a positive correlation in substitution rates between homologous X and homologous Y introns as well as between orthologous primate and perissodactyl introns. The male-to-female mutation rate ratio estimated from concatenated sequences of five perissodactyl introns was 3.88 (95% CI = 2.90-6.07). Using the data generated here and estimates available in the literature, we compared male mutation bias among several mammalian orders. We conclude that male mutation bias is significantly higher for organisms with long generation times (primates, perissodactyls, and felids) than for organisms with short generation times (e.g., rodents) since the former undergo a greater number of male germline cell divisions.

Synaptic plasticity is important for formation of long-term memories and in re-establishment of function following injury. Seven cDNAs enriched following lesion in the hippocampus of the rat have been isolated using a PCR-based cDNA suppression subtraction hybridization. Sequence analysis resulted in the identification of two genes with known roles in synaptic development and neuronal activities: astrotactin and calcineurin. These two neuron-specific genes have established roles in development or synaptogenesis. Sequence analysis of the other five additional genes shows that two are likely to be involved in G-protein signaling pathways, one is a WD repeat protein, and the remaining two are entirely novel. All seven candidates are expressed in the hippocampus and, in some cases, cortical layers of adult brains. RT-PCR data show that expression increases following synaptogenic lesion. Immunocytochemical analysis in primary hippocampal neurons showed that Calcineurin immunoreactivity was redistributed in neurons during 2 weeks in culture. This redistribution suggests that Calcineurin's role changes during neurite outgrowth immediately prior to synapse formation in vitro. In addition, inhibiting Calcineurin activity with cyclosporin A enhanced neurite outgrowth, suggesting that Calcineurin has a regulatory role in axon sprouting. The discovery of previously unknown genes involved in the response to neurodegeneration will contribute to our understanding of neural development, responses to CNS trauma, and neurodegenerative diseases.

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.

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 Treponema denticola surface protease complex, consisting of PrtP protease (dentilisin) and two auxiliary polypeptides (PrcA1 and PrcA2), is believed to contribute to periodontal disease by degrading extracellular matrix components and disrupting host intercellular signaling. Previously, we showed that transcription of the protease operon initiates upstream of TDE0760 (herein designated prcB ), the open reading frame immediately 5′ of prcA-prtP . The prcB gene is conserved in T. denticola strains. PrcB localizes to the detergent phase of Triton X-114 cell surface extracts and migrates as a 22-kDa polypeptide, in contrast to the predicted 17-kDa cytoplasmic protein encoded in the annotated T. denticola genome. Consistent with this observation, the PrcB N terminus is unavailable for Edman sequencing, suggesting that it is acylated. Nonpolar deletion of prcB in T. denticola showed that PrcB is required for production of PrtP protease activity, including native PrtP cleavage of PrcA to PrcA1 and PrcA2. A 6×His-tagged PrcB protein coimmunoprecipitates with native PrtP, using either anti-PrtP or anti-His-tag antibodies, and recombinant PrtP copurifies with PrcB-6×His in nickel affinity chromatography. Taken together, these data are consistent with identification of PrcB as a PrtP-binding lipoprotein that likely stabilizes the PrtP polypeptide during localization to the outer membrane.

Drosophila pseudoobscura harbors a rich polymorphism for paracentric inversions on the third chromosome, and the clines in the inversion frequencies across the southwestern United States indicate that strong natural selection operates on them. Isogenic inversion strains were made from isofemale lines collected from four localities, and eight molecular markers were mapped on the third chromosome. Nucleotide diversity was measured for these loci and formed the basis of an evolutionary genomic analysis. The loci were differentiated among inversions. The inversions did not show significant differences among populations, however, likely the result of extensive gene flow among populations. Some loci had significant reductions in nucleotide diversity within inversions compared with interspecies divergence, suggesting that these loci are near inversion breakpoints or are near targets of directional selection. Linkage disequilibrium (LD) levels tended to decrease with distance between loci, indicating that some genetic exchange occurs among gene arrangements despite the presence of inversions. In some cases, however, adjacent genes had low levels of interlocus LD and loosely linked genes had high levels of interlocus LD, suggesting strong epistatic selection. Our results support the hypothesis that the inversions of D. pseudoobscura have emerged as suppressors of recombination to maintain positive epistatic relationships among loci within gene arrangements that developed as the species adapted to a heterogeneous environment.