Characterization of Treponema denticola Major Surface Protein (Msp) by Deletion Analysis and Advanced Molecular Modeling.

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 ß-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.

Simultaneous Determination of Absolute Configuration and Quantity of Lipopeptides Using Chiral Liquid Chromatography/Mass Spectrometry and Diastereomeric Internal Standards.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

Bacterial flagellum: visualizing the complete machine in situ.

Electron tomography of frozen-hydrated bacteria, combined with single particle averaging, has produced stunning images of the intact bacterial flagellum, revealing features of the rotor, stator and export apparatus.

In situ structure of the complete Treponema primitia flagellar motor.

The bacterial flagellar motor is an amazing nanomachine: built from approximately 25 different proteins, it uses an electrochemical ion gradient to drive rotation at speeds of up to 300 Hz (refs 1, 2). The flagellar motor consists of a fixed, membrane-embedded, torque-generating stator and a typically bidirectional, spinning rotor that changes direction in response to chemotactic signals. Most structural analyses so far have targeted the purified rotor, and hence little is known about the stator and its interactions. Here we show, using electron cryotomography of whole cells, the in situ structure of the complete flagellar motor from the spirochaete Treponema primitia at 7 nm resolution. Twenty individual motor particles were computationally extracted from the reconstructions, aligned and then averaged. The stator assembly, revealed for the first time, possessed 16-fold symmetry and was connected directly to the rotor, C ring and a novel P-ring-like structure. The unusually large size of the motor suggested mechanisms for increasing torque and supported models wherein critical interactions occur atop the C ring, where our data suggest that both the carboxy-terminal and middle domains of FliG are found.

Phenol/water extract of Treponema socranskii subsp. socranskii as an antagonist of Toll-like receptor 4 signalling.

Treponema socranskii is one of the most frequently found oral spirochaetes in periodontitis and endodontic infections. LPS or glycolipids from bacteria are potent stimulators of innate immune and inflammatory systems. In this study the bioactivity of a phenol/water extract from T. socranskii subsp. socranskii (TSS-P) was analysed. TSS-P showed minimal endotoxicity and no inducing potential for proinflammatory cytokines (TNF-alpha and IL-8) or for intercellular adhesion molecule-1 (ICAM-1) in human monocyte cell line THP-1 cells and primary cultured human gingival fibroblasts. Rather, it inhibited ICAM-1 expression and IL-8 secretion from cells stimulated by the LPS of Escherichia coli and Actinobacillus actinomycetemcomitans, which are known to be Toll-like receptor 4 (TLR4) agonists. However, this antagonistic activity was not shown in cells stimulated by peptidoglycan or IL-1beta. As its antagonistic mechanism, TSS-P blocked the binding of E. coli LPS to LPS-binding protein (LBP) and CD14, which are molecules involved in the recruitment of LPS to the cell membrane receptor complex TLR4-MD-2 for the intracellular signalling of LPS. TSS-P itself did not bind to MD-2 or THP-1 cells, but inhibited the binding of E. coli LPS to MD-2 or to the cells in the presence of serum (which could be replaced by recombinant human LBP and recombinant human CD14). The results suggest that TSS-P acts as an antagonist of TLR4 signalling by interfering with the functioning of LBP/CD14.

Treponema berlinense sp. nov. and Treponema porcinum sp. nov., novel spirochaetes isolated from porcine faeces.

Limit-dilution procedures were used to isolate seven, helically coiled bacterial strains from faeces of swine that constituted two unidentified taxa. Comparative 16S rRNA gene sequence analysis showed highest similarity values with species of the genus Treponema indicating that the isolates are members of this genus. Strain 7CPL208(T), as well as five further isolates, and 14V28(T) displayed the highest 16S rRNA gene sequence similarities with Treponema pectinovorum ATCC 33768(T) (92.3%) and Treponema parvum OMZ 833(T) (89.9%), respectively. Polar lipid profiles distinguished 7CPL208(T) and 14V28(T) from each other as well as from related species. Based on their phenotypic and genotypic distinctiveness, strains 7CPL208(T) and 14V28(T) are suggested to represent two novel species of the genus Treponema, for which the names Treponema berlinense sp. nov. and Treponema porcinum sp. nov. are proposed. The type strain for Treponema berlinense is 7CPL208(T) (=ATCC BAA-909(T)=CIP 108244(T)=JCM 12341(T)) and for Treponema porcinum 14V28(T) (=ATCC BAA-908(T)=CIP 108245(T)=JCM 12342(T)).

Structure and function of an unusual family of protein phosphatases: the bacterial chemotaxis proteins CheC and CheX.

In bacterial chemotaxis, phosphorylated CheY levels control the sense of flagella rotation and thereby determine swimming behavior. In E. coli, CheY dephosphorylation by CheZ extinguishes the switching signal. But, instead of CheZ, many chemotactic bacteria contain CheC, CheD, and/or CheX. The crystal structures of T. maritima CheC and CheX reveal a common fold unlike that of any other known protein. Unlike CheC, CheX dimerizes via a continuous beta sheet between subunits. T. maritima CheC, as well as CheX, dephosphorylate CheY, although CheC requires binding of CheD to achieve the activity of CheX. Structural analyses identified one conserved active site in CheX and two in CheC; mutations therein reduce CheY-phosphatase activity, but only mutants of two invariant asparagine residues are completely inactive even in the presence of CheD. Our structures indicate that the flagellar switch components FliY and FliM resemble CheC more closely than CheX, but attribute phosphatase activity only to FliY.

Treponemal phospholipids inhibit innate immune responses induced by pathogen-associated molecular patterns.

Host innate immune responses to microbial components, known as pathogen-associated molecular patterns (PAMPs), are regulated and modified by cellular receptors and serum proteins, including Toll-like receptors (TLRs), CD14, and LPS-binding protein (LBP). We demonstrated that a treponemal membrane lipid inhibited PAMPs-induced immune responses. The chemical structure of the lipid was elucidated as a phosphatidylglycerol (PG) derivative, which is scarce in most mammalian tissues, but relatively abundant in treponemal membrane lipids. Natural and synthetic PG counterparts as well as related natural anionic phospholipids, phosphatidylinositol, phosphatidylserine, and cardiolipin, also demonstrated an inhibitory effect. Further, we noted that PG inhibited PAMPs-induced immune responses by blocking the binding of PAMPs with LBP and CD14. In addition, PG decreased proinflammatory cytokine production in serum of LPS-injected mice and depressed abscess formation in mice infected with treponemes. These results suggest that treponemal phospholipid interfere the function of LBP/CD14 and act as a modulator of innate immune responses.

Structural elucidation of polysaccharide part of glycoconjugate from Treponema medium ATCC 700293.

Glycoconjugates are distributed on the cell surfaces of some small-sized treponemes and have been reported to be completely different from lipopolysaccharides. We separated a glycoconjugate fraction from Treponema medium ATCC 700293, a medium-sized oral spirochete, to assess its immunobiological activities and elucidate the chemical structure of its polysaccharide part using phenol/water extraction, hydrophobic chromatography, and gel filtration. The glycoconjugate showed negligible or weak endotoxic and immunobiological properties. The chemical structure of the polysaccharide part was shown by two-dimensional NMR and MALDI-TOF-MS to be a tetrasaccharide backbone with two amino acids: [-->4)beta-d-GlcpNAc3NAcA(1-->4)beta-d-ManpNAc3NAOrn(1-->3)beta-d-GlcpNAc(1-->3)alpha-D-Fucp4NAsp(1-->] where GlcNAc3NAcA is 2,3-diacetamido-2,3-dideoxyglucuronic acid, ManNAc3NAOrn is Ndelta-(2-acetamido-3-amino-2,3-dideoxymannuronyl)ornithine, and Fuc4NAsp is 4-(alpha-aspartyl)amino-4,6-dideoxygalactose.

Cloning and characterization of a major surface protein (MspTL) of Treponema lecithinolyticum associated with rapidly progressive periodontitis.

The gene encoding a major surface protein (MspTL) of Treponema lecithinolyticum, a periodontopathogen, was cloned and sequenced. The mspTL gene has a 1770-bp open reading frame (ORF) encoding a protein of 590 amino acids with a predicted molecular mass of 65 kDa which had a typical prokaryotic signal sequence (19 amino acids). MspTL showed a high level of homology with major sheath protein (MspA) of Treponema maltophilum, phylogenetically the closest relative of T. lecithinolyticum. Southern blot analysis indicated that the mspTL gene exists in a single copy and Northern blot analysis showed that the mspTL transcript is monocistronic. Another ORF located downstream of mspTL was in the same orientation and encoded a putative protein, in which the first N-terminal 291 amino acids were identified. The homologous region of this protein is also a part on the T. maltophilum mspA locus.

Outer sheath associated proteins of the oral spirochete Treponema maltophilum.

We recently cloned the major outer membrane protein of Treponema maltophilum [Heuner, K., Choi, B.K., Schade, R., Moter, A., Otto, A., Göbel, U.B., J. Bacteriol. 181, 1025-1029]. Here we report the localization of the major sheath protein (Msp)A protein in T. maltophilum by immunogold electron microscopy and its expression. Northern blot analysis revealed that mspA is expressed constitutively as a monocistronic unit. The transcription initiation site of the mspA gene was identified by primer extension analysis. A further screening of a genomic library of T. maltophilum with an anti-outer membrane fraction antibody was done. We were able to clone DNA regions of T. maltophilum encoding putative sugar transport operons and putative outer membrane proteins of this oral treponeme which has a high prevalence in periodontal lesions.

Involvement of lipopolysaccharide binding protein, CD14, and Toll-like receptors in the initiation of innate immune responses by Treponema glycolipids.

Culture supernatants from Treponema maltophilum associated with periodontitis in humans and Treponema brennaborense found in a bovine cattle disease accompanied with cachexia caused a dose-dependent TNF-alpha synthesis in human monocytes increasing with culture time. This activity could be reduced significantly by blocking the CD14-part of the LPS receptor using the My 4 mAb and by polymyxin B. In the murine macrophage cell line RAW 264.7, Treponema culture supernatants induced TNF-alpha secretion in a LPS binding protein (LBP)-dependent fashion. To enrich for active compounds, supernatants were extracted with butanol, while whole cells were extracted using a phenol/water method resulting in recovery of material exhibiting a similar activity profile. An LPS-LBP binding competition assay revealed an interaction of the treponeme phenol/water extracts with LBP, while precipitation studies implied an affinity to polymyxin B and endotoxin neutralizing protein. Macrophages obtained from C3H/HeJ mice carrying a Toll-like receptor (TLR)-4 mutation were stimulated with treponeme extracts for NO release to assess the role of TLRs in cell activation. Furthermore, NF-kappaB translocation in TLR-2-negative Chinese hamster ovary (CHO) cells was studied. We found that phenol/water-extracts of the two strains use TLRs differently with T. brennaborense-stimulating cells in a TLR-4-dependent fashion, while T. maltophilum-mediated activation apparently involved TLR-2. These results indicate the presence of a novel class of glycolipids in Treponema initiating inflammatory responses involving LBP, CD14, and TLRs.

Crystal structure of cystalysin from Treponema denticola: a pyridoxal 5'-phosphate-dependent protein acting as a haemolytic enzyme.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

A spirochete isolated from a case of severe virulent ovine foot disease is closely related to a Treponeme isolated from human periodontitis and bovine digital dermatitis.

The isolation of spirochetes from severe ovine foot disease has been reported recently by our research group. In this study we describe the preliminary classification of this spirochete based on nucleotide sequence analysis of the PCR-amplified 16S rRNA gene. Phylogenetic analysis of this sequence in comparison with other previously reported 16S rRNA gene sequences showed that the spirochete belonged to the treponemal phylotype Treponema vincentii which has been associated with bovine digital dermatitis and human periodontal disease. Further work is required to define the common virulence determinants of these closely related treponemes in the aetiology of these tissue destructive diseases.

Identification of a Treponema denticola OppA homologue that binds host proteins present in the subgingival environment.

Proteins secreted or exported by Treponema denticola have been implicated as mediators of specific interactions between the spirochete and subgingival tissues in periodontal diseases. However, limited information is available on the ability of this peptidolytic organism to bind or transport soluble peptides present in the subgingival environment. A prominent 70-kDa protein was isolated from surface extracts of T. denticola ATCC 35405. A clone expressing a portion of the protein was identified in an Escherichia coli expression library of T. denticola DNA. DNA sequence analysis showed that the cloned gene encoded a peptide homologous to OppA, the solute binding protein of an ATP-binding cassette-type peptide transporter involved in peptide uptake and environmental signaling in a wide range of bacteria. Genes encoding OppB, -C, -D, and -F were identified directly downstream of oppA in T. denticola. OppA was present in representative strains of T. denticola and in Treponema vincentii but was not detected in Treponema pectinovorum or Treponema socranskii. Immunogold electron microscopy suggested that OppA was accessible to proteins at the surface of the spirochete. Native OppA bound soluble plasminogen and fibronectin but did not bind to immobilized substrates or epithelial cells. A T. denticola oppA mutant bound reduced amounts of soluble plasminogen, and plasminogen binding to the parent strain was inhibited by the lysine analog epsilon-aminocaproic acid. Binding of soluble host proteins by OppA may be important both for spirochete-host interactions in the subgingival environment and for uptake of peptide nutrients.

Fimbria-mediated coaggregation between human oral anaerobes Treponema medium and Porphyromonas gingivalis.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.

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

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

Isolation and chemical analysis of a lipopolysaccharide from the outer membrane of the oral anaerobic spirochete Treponema pectinovorum.

Isolation of a putative lipopolysaccharide from the surface of the oral treponeme, Treponema pectinovorum, revealed it to contain larger amounts of 3-deoxy-D-manno-octulosonic acid compared with other oral Treponema species. This molecule was isolated from the outer membrane of T. pectinovorum and had chemical characteristics of a putative lipopolysaccharide. The yield of lipopolysaccharide was between 0.6% and to 1.1% of the bacterial dry weight. The purified molecule was resistant to the action of proteinases and consisted of both sugars and lipids. 3-Deoxy-D-manno-octulosonic acid and hexoses accounted for 6.1-8.7% and 17.6-20.2%, respectively of the dry weight. Carbohydrate compositional analysis revealed the presence of glucose, galactose, 2-acetamido-2-deoxy-glucose, rhamnose and 6-deoxy-talose in the molar ratio of 1.00:0.96:0.19:0.88:0.98, respectively. No heptose was detected. The fatty acid analysis determined the presence of straight chain, C13:00, C14:00, C15:00 and C17:00 acids, as well as branched chain, C13:00, C14:00 and two species of C15:00, acids. Electrophoretic analysis indicated that the lipopolysaccharide was present as two major species.

The Treponema denticola major sheath protein is predominantly periplasmic and has only limited surface exposure.

The recent discovery that the Treponema pallidum genome encodes 12 orthologs of the Treponema denticola major sheath protein (Msp) prompted us to reexamine the cellular location and topology of the T. denticola polypeptide. Experiments initially were conducted to ascertain whether Msp forms an array on or within the T. denticola outer membrane. Transmission electron microscopy (EM) of negatively stained and ultrathin-sectioned organisms failed to identify a typical surface layer, whereas freeze-fracture EM revealed that the T. denticola outer membrane contains heterogeneous transmembrane proteins but no array. In contrast, a lattice-like structure was observed in vesicles released from mildly sonicated treponemes; combined EM and biochemical analyses demonstrated that this structure was the peptidoglycan sacculus. Immunoelectron microscopy (IEM) subsequently was performed to localize Msp in T. denticola. Examination of negatively stained whole mounts identified substantial amounts of Msp in sonicated organisms. IEM of ultrathin-sectioned, intact treponemes also demonstrated that the preponderance of antigen was unassociated with the outer membrane. Lastly, immunofluorescence analysis of treponemes embedded in agarose gel microdroplets revealed that only minor portions of Msp are surface exposed. Taken as a whole, our findings challenge the widely held belief that Msp forms an array within the T. denticola outer membrane and demonstrate, instead, that it is predominantly periplasmic with only limited surface exposure. These findings also have implications for our evolving understanding of the contribution(s) of Msp/Tpr orthologs to treponemal physiology and disease pathogenesis.

[Collagen-binding proteins of human oral spirochetes].

OBJECTIVE: To evaluate pathogenic effect of the collagen-binding proteins(CBP) of human oral spirochetes. METHODS: Immunoblot analysis, immunoelectron microscopy and other experiments had been used. RESULTS: It was showed that: type I, IV and V CBP were detected in heated and unheated preparations from Treponema denticola(Td) ATCC 33520 and Treponema socranskii (Ts) ATCC 35534. Few CBP, however, were detected in heated and unheated preparations from a recently characterized isloate, Treponema medium(Tm) strain G7201. 27,000 type V CBP of Td, 110,000 type I of Ts and 95,000 type IV of Tm were located on the envelopes of the individual cells. The adherence of Td to the collagen-coated surface was significantly greater than that of Tm. CONCLUSION: The CBP on the oral spirochetal cells play an important role in their adherence to collagen-rich connective tissues of the host.