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Tannerella serpentiformis is a health-associated Gram-negative oral anaerobe, while its closest phylogenetic relative is the periodontal pathogen Tannerella forsythia. The pathogen employs glycan mimicry through protein O-glycosylation, displaying a terminal nonulosonic acid aiding in evasion of host immune recognition. Like T. forsythia, T. serpentiformis cells are covered with a 2D-crystalline S-layer composed of two abundant S-layer glycoproteins-TssA and TssB. In this study, we elucidated the structure of the O-linked glycans of T. serpentiformis using 1D and 2D NMR spectroscopy analyzing S-layer glycopeptides and ß-eliminated glycans. We found that T. serpentiformis produces two highly fucosylated, branched glycoforms carrying non-carbohydrate modifications, with the structure [2-OMe-Fuc-(α1,2)]-4-OMe-Glc-(ß1,3)-[Fuc-(α1,4)]-2-NAc-GlcA-(ß1,4)-[3-NH2, 2,4-OMe-Fuc-(α1,3)]-Fuc-(α1,4)-Xyl-(ß1,4)-[3-OMe-Fuc-(α1,3)]-GlcA-(α1,2)-[Rha-(α1,4]-Gal, where the 3OMe-Fuc is variable; each glycoform contains a rare 2,4-methoxy, 3-amino-modified fucose. These glycoforms support the hypothesis that nonulosonic acid is a hallmark of pathogenic Tannerella species. A combined glycoproteomics and bioinformatics approach identified multiple sites within TssA (14 sites) and TssB (21 sites) to be O-glycosylated. LC-MS/MS confirmed the presence of the Bacteroidetes O-glycosylation motif (D)(S/T) (L/V/T/A/I) in Tannerella species, including the newly identified candidate "N" for the third position. Alphfold2 models of the S-layer glycoproteins were created revealing an almost uniform spatial distribution of the two glycoforms at the N-terminal two thirds of the proteins supported by glycoproteomics, with glycans facing outward. Glycoproteomics identified 921 unique glycopeptide sequences corresponding to 303 unique UniProt IDs. GO-term enrichment analysis versus the entire T. serpentiformis proteome classified these proteins as mainly membrane and cell periphery-associated glycoproteins, supporting a general protein O-glycosylation system in T. serpentiformis.

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Introduction: Degradation of host proteins by bacterial proteases leads to the subversion of the host response and disruption of oral epithelial integrity, which is considered an essential factor in the progression of periodontitis. High-temperature requirement A (HtrA) protease, which is critical for bacterial survival and environmental adaptation, is found in several oral bacteria, including the periodontal pathogen Tannerella forsythia. This study investigated the proteolytic activity of HtrA from T. forsythia and its ability to modulate the host response. Methods: HtrA of T. forsythia was identified bioinformatically and produced as a recombinant protein. T. forsythia mutants with depleted and restored HtrA production were constructed. The effect of T. forsythia wild-type, mutants and recombinant HtrA on the degradation of casein and E-cadherin was tested in vitro. Additionally, the responses of human gingival fibroblasts and U937 macrophages to the different HtrA-stimuli were investigated and compared to those triggered by the HtrA-deficient mutant. Results: T. forsythia wild-type producing HtrA as well as the recombinant enzyme exhibited proteolytic activity towards casein and E-cadherin. No cytotoxic effect of either the wild-type, T. forsythia mutants or rHtrA on the viability of host cells was found. In hGFB and U937 macrophages, both T. forsythia species induced an inflammatory response of similar magnitude, as indicated by gene and protein expression of interleukin (IL)-1ß, IL-6, IL-8, tumour necrosis factor α and monocyte chemoattractant protein (MCP)-1. Recombinant HtrA had no significant effect on the inflammatory response in hGFBs, whereas in U937 macrophages, it induced a transient inflammatory response at the early stage of infection. Conclusion: HtrA of T. forsythia exhibit proteolytic activity towards the host adhesion molecule E-cadherin and has the potential to influence the host response. Its role in the progression of periodontitis needs further clarification.

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OBJECTIVE: The red-complex bacteria Porphyromonas gingivalis and Tannerella forsythia together with Fusobacterium nucleatum are essential players in periodontitis. This study investigated the bacterial interplay with human periodontal ligament mesenchymal stromal cells (hPDL-MSCs) which act in the acute phase of periodontal infection. DESIGN: The capability of the bacteria to induce an inflammatory response as well as their viability, cellular adhesion and invasion were analyzed upon mono- and co-infections of hPDL-MSCs to delineate potential synergistic or antagonistic effects. The expression level and concentration of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1 were measured using qRT-PCR and ELISA. Viability, invasion, and adhesion were determined quantitatively using agar plate culture and qualitatively by confocal microscopy. RESULTS: Viability of P. gingivalis and T. forsythia but not F. nucleatum was preserved in the presence of hPDL-MSCs, even in an oxygenated environment. F. nucleatum significantly increased the expression and concentration of IL-6, IL-8 and MCP-1 in hPDL-MSCs, while T. forsythia and P. gingivalis caused only a minimal inflammatory response. Co-infections in different combinations had no effect on the inflammatory response. Moreover, P. gingivalis mitigated the increase in cytokine levels elicited by F. nucleatum. Both red-complex bacteria adhered to and invaded hPDL-MSCs in greater numbers than F. nucleatum, with only a minor effect of co-infections. CONCLUSIONS: Oral bacteria of different pathogenicity status interact differently with hPDL-MSCs. The data support P. gingivalis' capability to manipulate the inflammatory host response. Further research is necessary to obtain a comprehensive picture of the role of hPDL-MSCs in more complex oral biofilms.

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Streptococci are primary colonizers of the oral cavity where they are ubiquitously present and an integral part of the commensal oral biofilm microflora. The role oral streptococci play in the interaction with the host is ambivalent. On the one hand, they function as gatekeepers of homeostasis and are a prerequisite for the maintenance of oral health - they shape the oral microbiota, modulate the immune system to enable bacterial survival, and antagonize pathogenic species. On the other hand, also recognized pathogens, such as oral Streptococcus mutans and Streptococcus sobrinus, which trigger the onset of dental caries belong to the genus Streptococcus. In the context of periodontitis, oral streptococci as excellent initial biofilm formers have an accessory function, enabling late biofilm colonizers to inhabit gingival pockets and cause disease. The pathogenic potential of oral streptococci fully unfolds when their dissemination into the bloodstream occurs; streptococcal infection can cause extra-oral diseases, such as infective endocarditis and hemorrhagic stroke. In this review, the taxonomic diversity of oral streptococci, their role and prevalence in the oral cavity and their contribution to oral health and disease will be discussed, focusing on the virulence factors these species employ for interactions at the host interface.

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The oral cavity harbors a diverse and dynamic bacterial biofilm community which is pivotal to oral health maintenance and, if turning dysbiotic, can contribute to various diseases. Glycans as unsurpassed carriers of biological information are participating in underlying processes that shape oral health and disease. Bacterial glycoinfrastructure-encompassing compounds as diverse as glycoproteins, lipopolysaccharides (LPSs), cell wall glycopolymers, and exopolysaccharides-is well known to influence bacterial fitness, with direct effects on bacterial physiology, immunogenicity, lifestyle, and interaction and colonization capabilities. Thus, understanding oral bacterias' glycoinfrastructure and encoded glycolanguage is key to elucidating their pathogenicity mechanisms and developing targeted strategies for therapeutic intervention. Driven by their known immunological role, most research in oral glycobiology has been directed onto LPSs, whereas, recently, glycoproteins have been gaining increased interest. This review draws a multifaceted picture of the glycolanguage, with a focus on glycoproteins, manifested in prominent oral bacteria, such as streptococci, Porphyromonas gingivalis, Tannerella forsythia, and Fusobacterium nucleatum. We first define the characteristics of the different glycoconjugate classes and then summarize the current status of knowledge of the structural diversity of glycoconjugates produced by oral bacteria, describe governing biosynthetic pathways, and list biological roles of these energetically costly compounds. Additionally, we highlight emerging research on the unraveling impact of oral glycoinfrastructure on dental caries, periodontitis, and systemic conditions. By integrating current knowledge and identifying knowledge gaps, this review underscores the importance of studying the glycolanguage oral bacteria speak to advance our understanding of oral microbiology and develop novel antimicrobials.

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Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

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The recently identified bacterium Tannerella serpentiformis is the closest phylogenetic relative of Tannerella forsythia, whose presence in oral biofilms is associated with periodontitis. Conversely, T. serpentiformis is considered health-associated. This discrepancy was investigated in a comparative study of the two Tannerella species. The biofilm behavior was analyzed upon their addition and of Porphyromonas gingivalis-each bacterium separately or in combinations-to an in vitro five-species oral model biofilm. Biofilm composition and architecture was analyzed quantitatively using real-time PCR and qualitatively by fluorescence in situ hybridization/confocal laser scanning microscopy, and by scanning electron microscopy. The presence of T. serpentiformis led to a decrease of the total cell number of biofilm bacteria, while P. gingivalis was growth-promoting. This effect was mitigated by T. serpentiformis when added to the biofilm together with P. gingivalis. Notably, T. serpentiformis outcompeted T. forsythia numbers when the two species were simultaneously added to the biofilm compared to biofilms containing T. forsythia alone. Tannerella serpentiformis appeared evenly distributed throughout the multispecies biofilm, while T. forsythia was surface-located. Adhesion and invasion assays revealed that T. serpentiformis was significantly less effective in invading human gingival epithelial cells than T. forsythia. Furthermore, compared to T. forsythia, a higher immunostimulatory potential of human gingival fibroblasts and macrophages was revealed for T. serpentiformis, based on mRNA expression levels of the inflammatory mediators interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 and tumor necrosis factor α, and production of the corresponding proteins. Collectively, these data support the potential of T. serpentiformis to interfere with biological processes relevant to the establishment of periodontitis.

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Self-assembling (glyco)protein surface layers (S-layers) are ubiquitous prokaryotic cell-surface structures involved in structural maintenance, nutrient diffusion, host adhesion, virulence, and other processes, which makes them appealing targets for therapeutics and biotechnological applications as biosensors or drug delivery systems. However, unlocking this potential requires expanding our understanding of S-layer properties, especially the details of surface-attachment. S-layers of Gram-positive bacteria often are attached through the interaction of S-layer homology (SLH) domain trimers with peptidoglycan-linked secondary cell wall polymers (SCWPs). Cocrystal structures of the SLH domain trimer from the Paenibacillus alvei S-layer protein SpaA (SpaASLH) with synthetic, terminal SCWP disaccharide and trisaccharide analogs, together with isothermal titration calorimetry binding analyses, reveal that while SpaASLH accommodates longer biologically relevant SCWP ligands within both its primary (G2) and secondary (G1) binding sites, the terminal pyruvylated ManNAc moiety serves as the nearly exclusive SCWP anchoring point. Binding is accompanied by displacement of a flexible loop adjacent to the receptor site that enhances the complementarity between protein and ligand, including electrostatic complementarity with the terminal pyruvate moiety. Remarkably, binding of the pyruvylated monosaccharide SCWP fragment alone is sufficient to cause rearrangement of the receptor-binding sites in a manner necessary to accommodate longer SCWP fragments. The observation of multiple conformations in longer oligosaccharides bound to the protein, together with the demonstrated functionality of two of the three SCWP receptor-binding sites, reveals how the SpaASLH-SCWP interaction has evolved to accommodate longer SCWP ligands and alleviate the strain inherent to bacterial S-layer adhesion during growth and division.

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Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a ß-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium's cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s-1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s-1 were revealed. P. alvei CsaB was inactive on synthetic pNP-ß-d-ManNAc and ß-d-ManNAc-ß-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

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We examined the decisions made about transfer of patients with minor injuries, when telemedicine support was provided to the remote nurses in two different ways: the telemedicine doctor either used a video link, or a telephone call with viewing of digital X-ray images (a low resolution version of Picture Archiving and Communications Systems [PACS]). A quasi-randomized study design was used, with a panel of 20 emergency medicine doctors who independently reviewed previously-stored consultations using the two modalities. In total, 60 case reviews were conducted during five sessions, representing 33 different cases from the routine workload of Minor Treatment Centres in Scotland. More experienced doctors transferred fewer patients than less experienced doctors. The proportion of patients transferred was higher when PACS was used than when video was used in most of the cases. A mixed effects logistic regression model was fitted to the data. The estimated odds for patient transfer were 56% lower when video was used instead of PACS (odds ratio 0.44, 95% confidence interval 0.20, 0.93). Although the cost implications are not yet known, video support for local decision-making should remain the preferred method of telemedicine for minor injuries work.

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New technologies can change healthcare delivery. Cisco HealthPresence, an integrated platform that combines video, audio, and call center technology with medical information to create a virtual clinic experience, was piloted on emergency department patients. The aim was to assess primary care consultations. Patients were supported by an assistant. The doctor was remote from the patient and collected details and a recommended management plan. The same doctor re-examined the patient face-to-face. All patients completed a questionnaire about the experience. Key staff and a small sample of patients were interviewed. One hundred and five (N = 105) patients were included; 42% were given advice, 25% were prescribed analgesia, 26% were prescribed antibiotics, and 15% were x-rayed. There were early problems with the digital stethoscope. Doctors reported that the management plan changed in 7% of cases after seeing the patient. At least 90% of patients reported a positive experience. All patients and staff interviewed were positive. Staff found equipment to be valid and reliable; a concern was the inability to perform "hands on" examination. Telemedicine requires a change in the way of consulting and staff must be interested in using the technology to understand the differences. As one of the doctors said, "HealthPresence was a positive experience." Greater numbers would be required to validate key findings. As judged by clinicians, HealthPresence was successful and potentially safe for triage of unscheduled cases. Different types of staffing models need to be considered to ensure optimum use of health professionals. This study has shown that, despite some limitations, most HealthPresence consultations were found to be safe and appropriate. Further study of this consultation technology is required. HealthPresence has the potential to transform access to services for many patients, and to improve the effectiveness of delivery across a number of services.

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Access to health care in remote settings is becoming increasingly difficult in Scotland. We have investigated the feasibility of a telemedicine 'booth'. Two telemedicine booths were constructed for display at the Royal Highland Show in Edinburgh. One was equipped for patient use and one for the doctor. The booths contained videoconferencing and physiological monitoring equipment connected via an IP link at a bandwidth of 1.1 Mbit/s. The picture resolution was 4CIF (704 x 576 pixels). A total of 238 members of the public used the booth for a teleconsultation with a doctor. Ninety-three percent completed questionnaires. Of the 221 respondents, 75% saw the booth as an opportunity to access specialist advice; 84% felt that the booth would save them attending a hospital or clinic; 60% felt that it would improve the way they looked after their own health. The concept of a telemedicine booth appears both feasible and acceptable to the public.

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On any one day, approximately 15,000-28,000 oil industry personnel are employed in the North Sea oil and gas industry. First aid in this remote environment is provided by non-medical staff on the rigs. Remote specialist advice via videoconferencing should improve the quality of offshore health care and reduce avoidable medical evacuations. Satellite communications and videoconferencing equipment was installed on the 'Alwyn North' oil platform, with medical advice provided via a call centre in Milan. Over a nine-month period, trial telemedicine links were conducted approximately twice per week. The three onshore physicians were very satisfied on each occasion with communications and diagnostic data image quality, including the ultrasound screening carried out by the rig provider. Remote specialist advice via videoconferencing should reduce unnecessary and/or untimely patient evacuation to hospital or onshore for medical assessment.

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The Grampian Minor Injuries Telemedicine Service has been operating since 2001 supporting 15 minor injury units (MIUs) in community hospitals. Currently over 120 new patients are seen each month. We conducted a retrospective review to estimate the number of patients who were sent to the main hospital emergency department (ED) who would have been suitable for telemedicine treatment instead. All attendances at three MIUs and onward referrals to the ED during the months January and July 2006 were identified from a database. A total of 112 patients were referred from the three MIUs during the study period. MIU C, which utilized teleconsultations the most, referred the lowest proportion of its patients (2%). MIU B, which had all X-rays reviewed by a general practitioner, referred the most (85%). At MIU B, 80-85% of patients referred to the ED without having a teleconsultation could have been managed by telemedicine. Telemedicine for MIUs has been repeatedly reported in the medical literature as being successful, but widespread usage of this technique remains to be achieved.

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A telemedicine service was established for the Scottish Police College with medical advice provided from the Aberdeen Royal Infirmary. Use of the service from November 2004 to February 2006 was audited. A total of 192 patients presented to the service during the 16-month study period. There were 97 teleconsultations. The remaining 95 patients (49%) were not seen by telemedicine due to technical problems. The complaints dealt with were 68% injuries and 32% minor illness (n = 97). The outcome of the teleconsultation was: advice alone 76%; referral to a general practitioner 10%; and A&E referral 14% (n = 97). The treatment advised was: over-the-counter medicine 85%; prescription 5%; and nil 10% (n = 97). Even with a 24% connection rate, the service overall was perceived as beneficial and the Police College wished to continue to use the service, if the technology difficulties could be resolved.

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A telemedicine service consisting of electrocardiogram (ECG) interpretation and advice on the management of chest pain offshore was offered to oil rig installations in the North Sea. A total of 14 subscribing oil rigs were supplied with thrombolytic drugs and rig paramedics were trained in their delivery. Electrocardiographs could be recorded using a standard ECG machine and then digitized using a scanner for transmission as an email attachment. Several oil companies invested in ECG equipment which allowed direct recording of the patient's ECG in electronic form for transmission by email. Uptake of the telemedicine service was very rapid. The majority of rig medics found the system very easy to use. During a 36-month study period from August 2004, 47 cases of chest pain were dealt with by telemedicine. Of these 47 cases, only six patients (13%) were airlifted to shore. The 41 remaining ECGs did not to have acute changes requiring immediate evacuation. The use of email for ECG transmission proved to be highly effective in managing chest pain offshore.

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