David Dymock

A higher plant seven-transmembrane receptor that influences sensitivity to cytokinins.

Abstract Background: All organisms perceive and respond to a profusion of environmental and endogenous signals that influence growth, development and behaviour. The G-protein signalling pathway is a highly conserved mechanism for transducing extracellular signals, and the superfamily of receptors that have seven transmembrane (7TM) domains is a primary element of this pathway. Evidence that heterotrimeric G proteins are involved in signal transduction in plants is accumulating, prompting speculation that plant 7TM receptors might exist. Results: Using information in the dbEST database of expressed sequence tags, we isolated an Arabidopsis thaliana gene ( GCR1 ) that encodes a protein with seven predicted membrane-spanning domains and other features characteristic of 7TM receptors. The protein shows 18–23% amino-acid identity (46–53% similarity) to, and good colinear alignment with, 7TM receptors from three different families. Its highest sequence identity is with the Dictyostelium cAMP receptors. GCR1 is expressed at very low levels in the roots, stems and leaves of Arabidopsis; it is a single-copy gene which maps close to the restriction fragment length polymorphism marker m291 on chromosome 5. Transgenic Arabidopsis expressing antisense GCR1 under the control of the constitutive cauliflower mosaic virus 35S promoter have reduced sensitivity to cytokinins in roots and shoots, yet respond normally to all other plant hormones. This suggests a functional role for GCR1 in cytokinin signal transduction. Conclusions: GCR1 encodes the first 7TM receptor homologue identified in higher plants and is involved in cytokinin signal transduction. This discovery suggests that 7TM receptors are ancient and predate the divergence of plants and animals.

Corrosion of orthodontic appliances—should we care?

Contemporary orthodontics relies on various bonded attachments, archwires, and other devices to achieve tooth movement. These components are composed of varying materials with their own distinctive physical and mechanical properties. The demands made on them are complex because they are placed under many stresses in the oral environment. These include immersion in saliva and ingested fluids, temperature fluctuations, and masticatory and appliance loading. The combination of these materials in close proximity and in hostile conditions can result in corrosion. Our purpose in this article was to consider the literature to date with regard to potential mechanical, clinical, and health implications of orthodontic corrosion.

Binding Properties and Adhesion-Mediating Regions of the Major Sheath Protein of Treponema denticola ATCC 35405

ABSTRACT There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type I, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Molecular detection of novel anaerobic species in dentoalveolar abscesses.

The ability to isolate in pure culture the bacteria that cause 1492R [2]. The amplified genes were singularized by cloning, reamplified with the same primers, and then together with amplidisease in humans has been a cornerstone of the advances made in medical microbiology over the past hundred years and an essential fied 16S rRNA genes from the cultivated bacteria were digested component of Koch’s postulates. The etiologic agents of the majorwith a range of restriction endonucleases. ity of the diseases suspected to be caused by bacteria have been The fragments were separated by electrophoresis to give restricdiscovered by this method. However, the advances made as a result tion-fragment-length polymorphism (RFLP) profiles, which were of the use of artificial culture media have perhaps been fortuitous used to group the cloned genes. Members of a group shared the because our knowledge of the microorganisms found on earth is same RFLP profiles for at least three restriction endonucleases. extremely limited; it has been estimated that õ1% of the bacteria The RFLP profiles from the cultivated isolates were then compared on earth have been cultured. It is also estimated that 50% of to those of the groups of cloned genes. Representative clones of the oral flora is unculturable, and the proportion of unculturable groups exhibiting profiles that did not match any of the cultivable organisms at other body sites remains unknown. organisms were sequenced. In recent years studies of the microbial composition of biomass Sequences were identified by means of Similarity-Rank software found at environmental sites have been facilitated by advances in [3]. The sequences were then aligned to related 16S rRNA molecular biology. More specifically, PCR has enabled the amplisequences, and phylogenetic analysis was performed with the fication of specific genes, and the development of automated DNA PHYLIP suite of programs [4]. sequencing methods has allowed the rapid characterization of both The bacteria isolated from two of the samples are shown in entire genomes and specific genes in large numbers of organisms. table 1. A similar range of organisms was recovered from both Genes encoding ribosomal RNA have been found to be particularly samples, and both the identity and numbers of the species isolated useful for the construction of phylogenetic trees reflecting the evowere typical. For each sample, there were three groups of cloned lution of bacteria [1]. This is because these genes have been con16S rRNA genes that did not match with the profiles of the cultured served throughout evolution because of the need to preserve funcorganisms. The results of the sequence analysis for these groups tion. are shown in table 2. Some regions are highly conserved, allowing the design of uniGroups 3.4 and 3.5 showed high similarity to Prevotella oris and versal PCR primers for the amplification of the gene, while other Porphyromonas endodontalis, respectively. P. oris was isolated in regions are more variable, with sufficient information present to the study, but the cultivated organism exhibited a different RFLP differentiate at the species level. The 16S rRNA gene has been profile to that of the cloned group. Group 3.6 showed only low found to be particularly useful because its size of Ç1,500 bases similarity to Prevotella oralis. Further phylogenetic analysis of is sufficient to generate meaningful information, while it is short this sequence indicated that this group may represent a new genus enough for easy sequencing. Thus, 16S rRNA genes have been related to Bacteroides and Prevotella. Group 9.2 was identified as amplified from a variety of sources, including the environment P. endodontalis, while 9.3 was found to show a similarity of 93.6% and human infections, and then sequenced to reveal the diversity to Prevotella buccae. This level of similarity is indicative of the of organisms present at the site. group representing a new species of Prevotella. Dentoalveolar abscesses are acute infections associated with the Group 9.4 showed 89.7% similarity to Eubacterium brachy. The teeth, arising from pulpal infection secondary to dental caries. oral asaccharolytic Eubacterium species are a heterogeneous group Cultural analyses have revealed they are associated with a reof organisms currently undergoing reclassification [5]; all of the stricted group of organisms, principally members of the genera currently recognized species are likely to be elevated to genus rank Fusobacterium, Peptostreptococcus, Prevotella, and Streptococcus. In the studies described here, we adapted methods for the detection of uncultivable bacteria in the environment to the analysis of the microflora associated with dentoalveolar abscesses. Table 1. Bacteria cultured from pus from dentoalveolar abscesses. Pus was aspirated from dentoalveolar abscesses by needle and

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

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

Host collagen signal induces antigen I/II adhesin and invasin gene expression in oral Streptococcus gordonii

Microbial interactions with host molecules, and programmed responses to host environmental stimuli, are critical for colonization and initiation of pathogenesis. Bacteria of the genus Streptococcus are primary colonizers of the human mouth. They express multiple cell‐surface adhesins that bind salivary components and other oral bacteria and enable the development of polymicrobial biofilms associated with tooth decay and periodontal disease. However, the mechanisms by which streptococci invade dentine to infect the tooth pulp and periapical tissues are poorly understood. Here we show that production of the antigen I/II (AgI/II) family polypeptide adhesin and invasin SspA in Streptococcus gordonii is specifically upregulated in response to a collagen type I signal, minimally the tri‐peptide Gly‐Pro‐Xaa (where Xaa is hydroxyproline or alanine). Increased AgI/II polypeptide expression promotes bacterial adhesion and extended growth of streptococcal cell chains along collagen type I fibrils that are characteristically found within dentinal tubules. These observations define a new model of host matrix signal‐induced tissue penetration by bacteria and open the way for novel therapy opportunities for oral invasive diseases.

Axenic Culture of a Candidate Division TM7 Bacterium from the Human Oral Cavity and Biofilm Interactions with Other Oral Bacteria

ABSTRACT The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 μm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.

Treponema denticola chymotrypsin-like proteinase (CTLP) integrates spirochaetes within oral microbial communities

Treponema denticola is found ubiquitously in the human oral cavity and is mainly associated with bacterial communities implicated in the establishment and development of periodontal disease. The ability to become integrated within biofilm communities is crucial to the growth and survival of oral bacteria, and involves inter-bacterial coaggregation, metabolic cooperation, and synergy against host defences. In this article we show that the chymotrypsin-like proteinase (CTLP), found within a high-molecular-mass complex on the cell surface, mediates adherence of T. denticola to other potential periodontal pathogens, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia and Parvimonas micra. Proteolytic activity per se did not appear to be required for the interactions, and expression of the major outer-sheath protein (Msp) was not necessary, except for binding Parv. micra. Biofilms of densely packed cells and matrix, up to 40 µm in depth, were formed between T. denticola and P. gingivalis on salivary pellicle, with T. denticola cells enriched in the upper layers. Expression of CTLP, but not Msp, was critical for dual-species biofilm formation with P. gingivalis. T. denticola did not form dual-species biofilms with any of the other three periodontal bacterial species under various conditions. Synergy between T. denticola and P. gingivalis was also shown by increased inhibition of blood clotting, which was CTLP-dependent. The results demonstrate the critical role of CTLP in interactions of T. denticola with other oral micro-organisms, leading to synergy in microbial community development and host tissue pathogenesis.

Characterization of a novel family of fibronectin‐binding proteins with M23 peptidase domains from Treponema denticola

Interactions with fibronectin are important in the virulence strategies of a range of disease-related bacteria. The periodontitis-associated oral spirochaete Treponema denticola expresses at least two fibronectin-binding proteins, designated Msp (major surface protein) and OppA (oligopeptide-binding protein homologue). To identify other T. denticola outer membrane fibronectin-binding proteins, the amino acid sequence of the Treponema pallidum fibronectin-binding protein Tp0155 was used to survey the T. denticola genome. Seven T. denticola genes encoding orthologous proteins were identified. All but two were expressed in Escherichia coli and purified recombinant proteins bound fibronectin. Using antibodies to the N-terminal region of Tp0155, it was demonstrated that T. denticola TDE2318, with highest homology to Tp0155, was cell surface localized. Like Tp0155, the seven T. denticola proteins contained an M23 peptidase domain and four (TDE2318, TDE2753, TDE1738, TDE1297) contained one or two LysM domains. M23 peptidases can degrade peptidoglycan whereas LysM domains recognize carbohydrate polymers. In addition, TDE1738 may act as a bacteriocin based on homology with other bacterial lysins and the presence of an adjacent gene encoding a putative immunity factor. Collectively, these results suggest that T. denticola expresses fibronectin-binding proteins associated with the cell surface that may also have cell wall modifying or lytic functions.

Rapid differentiation of Prevotella intermedia and P. nigrescens by 16S rDNA PCR-RFLP

Prevotella intermedia and the newly described P. nigrescens cannot be reliably distinguished by phenotypic tests. In this study, restriction endonuclease digestion of amplified 16S rDNA (16S rDNA PCR-RFLP) was used to generate restriction profiles of the type strains of P. intermedia and P. nigrescens and 43 fresh isolates identified as belonging to one of the two species. Whole-cell protein profiles were obtained by SDS-PAGE for comparative purposes. The type strains of P. intermedia and P. nigrescens were easily distinguished by 16S rDNA PCR-RFLP and the fresh isolates were assigned to either species on the basis of their restriction profiles. The identifications obtained were identical to those obtained by protein profiles. 16S rDNA PCR-RFLP is a rapid and reliable method for the differentiation of P. intermedia and P. nigrescens.