Dental plaque forms sequentially, with Fusobacterium nucleatum facilitating the adhesion of pathogenic late colonizers. We hypothesize that a single-chain variable fragment (scFv) antibody library will enable the identification of F. nucleatum adhesins and help elucidate the molecular mechanisms of coaggregation between F. nucleatum and other bacteria. A 4X10^8 clones scFv phage display library was created using spleen RNA from a mouse immunized with F. nucleatum. The library was enriched by biopanning against F. nucleatum 6 times and 292 individual clones tested by ELISA reacted strongly to F. nucleatum. Sixty-two of those clones inhibited F. nucleatum coaggregation with Streptococcus sanguinus. Analysis of select clones revealed differences in coaggregation inhibition, recognition of outer membrane proteins, and BstOI restriction pattern. DNA sequencing showed 6 unique scFvs and of them 3 strongly inhibited interaction with 5 Streptococcus species. These scFvs recognize the outer membrane autotransporter protein RadD (Fn1526), as determined by mass spectrometry. / Farhan Khan placed second in the International Association for Dental Research/Unilever Hatton Competition in the Senior Basic Science Research Category representing Canada, while presenting the research contained in this dissertation. This international competition took place during the 90th General Session & Exhibition of the International Association for Dental Research in Iguaçu Falls, Brazil in June 2012.

Indiana University-Purdue University Indianapolis (IUPUI) / Periodontal disease is the leading cause of tooth loss in adults. Bacterial biofilm on tooth surfaces is the primary initiator of periodontal disease. Various factors contribute to the severity of periodontal disease including the different virulence factors of the bacteria within the biofilm. In the progression of periodontal disease, the microflora evolves from a predominantly Gram positive microbial population to a mainly Gram negative population. Specific gram negative bacteria with pronounced virulence factors have been implicated in the etiology and pathogenesis of periodontal disease, namely Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola which form the red complex of bacteria. The orange complex bacteria become more dominant in the maturation process of dental plaque and act to bridge the early colonizers of plaque with the later more dominant red complex bacterial and consists of such bacteria as Campylobacter showae, Campylobacter rectus, Fusobacterium nucleatum and Prevotella intermedia. Perhaps the most investigated contributing factor is the relationship between smoking and periodontal disease. When examining the association between cigarette smoking and interproximal bone loss, greater bone loss is associated with higher cigarette consumption, longer duration (i.e., pack year history) and higher lifetime exposure. The presence of various virulence factors such as the production of a capsular material, as well as the proteolytic activity of the various periopathodontic bacteria has been associated with the pathogenesis of periodontitis. Even though many different enzymes are produced in large quantities by these periodontal bacteria, trypsin-like enzymes, chymotrypsin-like enzymes and elastase-like enzymes, as well as dipeptidyl peptidase-like enzymes, have been thought to increase the destructive potential of the bacterium and mediate destruction of the periodontal apparatus. More specifically, it is hypothesized that the proteolytic activity of other clinically important periodontal pathogens, such as Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas assacharolyticus, is increased in the presence of nicotine. The purpose of this study was to determine the effects of nicotine on F. nucleatum, P. intermedia and P. assacharolyticus proteolytic activity. Cultures were maintained on anaerobic blood agar plates containing 3% sheep blood. Bacterial cells were harvested from the plates and washed. Washed F. nucleatum, P. intermedia and P. assacharolyticus cells were incubated with 1 mg/ml of nicotine. Bacterial cells not incubated with nicotine were used as positive controls. Secreted enzymatic activity was measured using the synthetic chromogenic substrates glycyl-L-proline-p-nitroanilide (GPPNA), N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide (SAAAPNA), N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (SAAPPPNA) and N-α-benzoyl-L-arginine-p-nitroanilide (L-BAPNA) (Sigma-Aldrich Products, St. Louis, MO, USA). Appropriate means and standard deviations were determined for each of the enzymatic activities measured and analysis of variance (ANOVA) was used to compare the groups utilizing a 5% significance level for all comparisons. Results demonstrated that after 60 minutes of incubation of F. nucleatum, P. intermedia and P. assacharolyticus cells with 1 mg/ml of nicotine and the various synthetic substrates, had the following proteolytic activity for GPPNA: 0.83 ± 0.14, 0.72 ± 0.03 and 0.67 ± 0.10, respectively; SAAAPNA: 0.82 ± 0.06, 0.76 ± 0.05 and 0.68 ± 0.08, respectively; SAAPPPNA: 0.90 ± 0.13, 0.85 ± 0.17 and 0.72 ± 0.03, respectively; and BAPNA: 0.81 ± 0.15, 0.74 ± 0.13 and 0.74 ± 0.16, respectively. In conclusion, the results indicate that in the presence of 1 mg/ml of nicotine, the proteolytic activity of F. nucleatum and P. assacharolyticus was increased with all of the synthetic substrates (with statistical significance seen only in the increases with F. nucleatum and GPPNA, SAAAPNA and BAPNA). The proteolytic activity exhibited an increasing trend in activity for P. intermedia with SAAPPPNA and BAPNA but a decreasing trend in activity with GPPNA and SAAAPNA when incubated with 1 mg/ml of nicotine, once again demonstrating no statistical significance for any of the substrates. Therefore, it could be concluded that based on these results nicotine at a concentration of 1 mg/ml may increase the proteolytic activity of periodontal pathogens and thus may increase periodontal disease activity and subsequent periodontal breakdown. Further studies are needed to validate these results utilizing different concentrations of nicotine.

Fusobacterium nucleatum is a common oral opportunistic bacterium that can cause different infections. In recent years, studies have shown that F. nucleatum is enriched in lesions in periodontal diseases, halitosis, dental pulp infection, oral cancer, and systemic diseases. Hence, it can promote the development and/or progression of these conditions. The current study aimed to assess research progress in the epidemiological evidence, possible pathogenic mechanisms, and treatment methods of F. nucleatum in oral and systemic diseases. Novel viewpoints obtained in recent studies can provide knowledge about the role of F. nucleatum in hosts and a basis for identifying new methods for the diagnosis and treatment of F. nucleatum-related diseases.

<p dir="ltr">The Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report highlights more than 2.8 million antibiotic infections each year, with at least 35,000 deaths per annum attributed to antibiotic resistance. The CDC’s 2022 COVID-19 Impact Report emphasizes a 15% increase in hospital-acquired resistant infections between 2019 and 2020, many which are caused by Gram-negative bacteria, bacteria characterized by two encapsulating membranes. The limited treatment options for Gram-negative bacterial infections underscore the critical need for new strategies to combat these pathogens. The β-barrel assembly machinery complex (BAM) is a protein complex located in the outer membrane (OM) of Gram-negative bacteria, facilitating the folding and insertion of β-barrel outer membrane proteins (OMPs) into the OM. Inhibiting the function of this complex is lethal for Gram-negative bacteria, making BAM a significant and promising drug target.</p><p dir="ltr"><i>Fusobacterium nucleatum</i> is a Gram-negative pathogen that functions in the oral microbiome, interacting with multiple levels of biofilm colonizers. <i>F. nucleatum</i> causes oral infections and is linked to colorectal cancer, impacting treatment response and disease recurrence. The pathogenicity of <i>F. nucleatum</i> in both biofilm formation and in cancer involves OMPs whose biogenesis relies on BAM; however, BAM has not been characterized in this organism. The goal of our study here is to better understand the composition, structure, and function of BAM and its potential as a drug target for <i>F. nucleatum</i>. We first used bioinformatics analysis and proteomics to investigate the putative composition of the BAM complex in <i>F. nucleatum</i>. While the core component BamA was identified, there was a notable absence of other typical accessory proteins in this organism's genome. Therefore, we postulate that unlike other bacteria such as <i>E. coli</i> and <i>A. baumannii</i>, the biogenesis of OMPs in <i>F. nucleatum</i> is mediated solely by BamA without the need of accessory components.</p><p dir="ltr">To investigate how BamA can accomplish OMP biogenesis itself, we employed biophysical techniques to analyze the structure of <i>Fn</i>BamA. We resolved the cryo-EM structure of <i>Fn</i>BamA in complex with several Fabs which showed novel structural features not previously observed in bacteria. In these structures, <i>Fn</i>BamA was found to contain four N-terminal POTRA domains arranged in a J-shaped conformation, rather than elongated. The Fab was found to bind primarily along POTRA 3 which likely stabilizes the unique conformation of the POTRA domains. The C-terminal 16-stranded b-barrel domain was observed as an inverted dimer, with the dimer interface mediated by direct interaction of the b1 strands along the lateral seam of both barrel domains. Additionally, we determined the X-ray crystal structure of the barrel domain alone which was found as a monomer. Measurements of the barrel domain of <i>Fn</i>BamA reveal it has a different shape and size than is found in other BamA structures such as in <i>E. coli</i>. Together, these structural differences provide clues to how <i>Fn</i>BamA alone may accomplish OMP biogenesis when additional components are required in other bacteria. Our ongoing studies aim to further characterize the molecular structure and function of <i>Fn</i>BamA in conjunction with promising antibiotics and other putative BAM components if discovered.</p>

<p>  </p> <p><em>Fusobacterium necrophorum</em> is a Gram-negative, anaerobic, opportunistic pathogen that causes necrotic infections in cattle leading to liver abscess, foot rot, and calf diphtheria. Particularly, liver abscess in cattle is reported at 20.7% annually, and leads to liver condemnation and an annual economic burden of about 62 million dollars to the feedlot industry. Antibiotic administration is the mainstay of treating these infections, but antibiotic resistance is unavoidable and demand for antibiotic-free, natural, and organic beef has demanded alternative therapies and preventatives. Vaccination is one of the best alternatives to prophylactic antibiotic administration. In this study, we have explored outer membrane proteins (OMPs) and outer membrane vesicles (OMVs) for potential vaccine candidates. OMPs and OMVs are vaccine targets because of their antigenic properties and host specificity. Additionally, we performed comparative genomic analysis of <em>F. necrophorum</em> species to identify additional virulence genes with vaccine potential, unique to the <em>F. necrophorum</em> and its virulent subspecies <em>necrophorum</em>. </p> <p>Protein- protein interaction investigation through binding assay and pulldown assay identified novel OMPs, namely 17kDa, 22kDa, and 66.3 kDa proteins, which were further characterized as OmpH, OmpA and Cell Surface Protein (CSP), respectively. In this study, these novel OMPs including previously characterized 43kDa OMPs were cloned, and recombinant proteins were expressed and purified. These recombinant proteins were used to generate polyclonal antibodies in rabbits, and their efficacy was studied using <em>in vitro</em> adhesion inhibition assays. The combination of two or more antibodies raised against the recombinant OMPs was significantly effective in reducing/neutralizing bacterial binding to bovine endothelial cells compared to individual antibody treatment. This suggests that a multiple subunit vaccine could be effective and provide sufficient evidence to perform <em>in vivo</em> studies. </p> <p>Similarly, we purified OMVs of <em>F. necrophorum</em> subspecies <em>necrophorum</em> 8L1 and analyzed its content using proteomics and lipidomics. Out of 342 proteins identified by tandem liquid chromatography mass spectrometry (LC-MS), OMPs and toxins were the most abundant. These included OMPs and toxins namely, 43 kDa OMP, OmpH, OmpA, CSP, FadA, leukotoxin family filamentous adhesin, N-terminal domain of hemagglutinin and other OMP transport and assembly factor protein. The presence of a subset of these proteins was further confirmed by western blot analysis. Lipidomics analysis showed that OMVs contained phospholipid, sphingolipid, and acetyl carnitine as the main lipid contents. Cytotoxicity assay on BL-3 cell line showed that these OMVs have a toxic effect on host immune cells and could impart immunomodulatory effect. All these findings suggest the vaccine potential of OMVs and demand dose-based <em>in vivo</em> study.</p> <p>In addition, we identified and characterized 5 clinical isolates of <em>F. necrophorum</em> using comparative genomics, UBCG (Up-to-date Bacterial Core Gene) based analysis enabled phylogenetic characterization of 46 <em>F. necrophorum</em> genomes into subspecies specific clades. The pangenome and recombination analysis showed the extensive disparity in accessory genes resulting in species divergence. Strikingly, we detected antimicrobial resistance gene for macrolides and tetracycline in one strain of <em>F. necrophorum</em>, a harbinger of the start of resistance and necessitating search for an alternative prophylactic method. We also noted common virulence genes, including toxins, outer membrane adhesion proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters and transporter proteins in <em>F. necrophorum</em> strains. A focused study on these genes could help identify the main genes of virulence and inform effective vaccination strategies against fusobacterial infections. </p> <p>Overall, the studies suggest adhesins and toxin and/or OMV-based subunit vaccine could be potential targets for vaccine development against fusobacterial infections.  </p>

Yes / Infection is the major reason for guided tissue regeneration/guided bone regeneration (GTR/GBR) membrane failure in clinical application. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection by electrospinning of poly(ε-caprolactone) (PCL) and gelatin blended with metronidazole (MNA). Acetic acid (HAc) was introduced to improve the miscibility of PCL and gelatin to fabricate homogeneous hybrid nanofiber membranes. The effects of the addition of HAc and the MNA content (0, 1, 5, 10, 20, 30, and 40 wt.% of polymer) on the properties of the membranes were investigated. The membranes showed good mechanical properties, appropriate biodegradation rate and barrier function. The controlled and sustained release of MNA from the membranes significantly prevented the colonization of anaerobic bacteria. Cells could adhere to and proliferate on the membranes without cytotoxicity until the MNA content reached 30%. Subcutaneous implantation in rabbits for 8 months demonstrated that MNA-loaded membranes evoked a less severe inflammatory response depending on the dose of MNA than bare membranes. The biodegradation time of the membranes was appropriate for tissue regeneration. These results indicated the potential for using MNA-loaded PCL/gelatin electrospun membranes as anti-infective GTR/GBR membranes to optimize clinical application of GTR/GBR strategies.

CONTEXTE : Le cancer colorectal (CCR) reste l’un des cancers les plus diagnostiqués au Canada et dans le monde. Malgré les efforts en matière de dépistage, la moitié des cancers colorectaux sont encore diagnostiqués à des stades avancés de la maladie, réduisant ainsi les chances de survie à ce cancer. La prévention du CCR reste encore primordiale et les efforts doivent être maintenus pour l’identification de ses facteurs de risque modifiables. Durant cette dernière décennie, on a assisté à un cumul d’évidence sur l’association entre l’enrichissement colorectal en une bactérie spécifique, Fusobacterium nucleatum (F. nucleatum), et le CCR. Ce qui a ressuscité le débat sur l’association même entre la maladie parodontale (MP) et le CCR, puisque F. nucleatum est non seulement une bactérie commensale de la bouche, mais surtout l’une des principaux agents pathogènes de la MP. Des études épidémiologiques antérieures avaient en effet déjà suggéré que le la MP serait associée au risque de CCR, mais leurs résultats sont restés non concluants à cause de leurs défaillances méthodologiques. La diffusion des bactéries parodontopathogènes, comme F. nucleatum, et de leurs produits de virulence de la bouche vers le côlon et la libération des médiateurs d’inflammation chronique dans la circulation générale sont les mécanismes incriminés dans l’association entre la MP et le CCR. OBJECTIFS: 1) Vérifier l’association entre la MP et le CCR; 2) Synthétiser la littérature sur l’association entre la détection de la bactérie F. nucleatum dans le côlon et le CCR et; 3) Vérifier la faisabilité d’investiguer l’expression concomitante de F. nucleatum, dans les deux milieux buccal et colorectal, en présence de lésions néoplasiques colorectales. Ces deux derniers objectifs sont un préambule à l’investigation du rôle des bactéries causales de la MP, précisément F. nucleatum, comme mécanisme d’association entre la MP et le CCR. MÉTHODES: Nous avons entrepris trois projets de recherche distincts qui répondent aux trois objectifs respectifs de recherche cités ci-dessus: 1) Une étude cas-témoins à base populationnelle (Projet COLDENT) a inclut 348 cas incidents de CCR et 310 témoins qui lui sont appariés dans des catégories d’âge et de sexe. Des données ont été collectées sur l’exposition à la MP et aux principaux facteurs de risque du CCR; 2) Une revue systématique avec méta-analyse d’études observationnelles ayant investigué la bactérie, F. nucleatum, dans des échantillons colorectaux, chez des cas de CCR et des témoins exempts de lésions cancéreuses ou précancéreuses colorectales; 6 et 3) Une étude pilote cas-témoins à base hospitalière, auprès de 22 cas de néoplasies colorectales et 21 témoins, examinés par coloscopie. Des échantillons de salive et des biopsies de muqueuse colorectale, ainsi que des données sur l’exposition à la MP et aux principaux facteurs de risque du CCR ont été collectées. RÉSULTATS: Les résultats de notre étude cas-témoins populationnelle suggèrent que le taux d’incidence de CCR était augmenté chez les personnes avec un historique positif de MP comparé aux personnes sans historique de MP, en ajustant pour la majorité des facteurs de confusion potentiels (Rapport des taux ajusté = 1,45 ; Intervalle de confiance (IC) à 95 % : 1,04-2,01). Les résultats de notre revue systématique incluant 24 études observationnelles ont montré que dans la majorité des études où la charge de F. nucleatum dans les échantillons colorectaux était quantifiée, celle-ci était significativement plus élevée chez les cas de CCR que chez les témoins. Les résultats de la méta-analyse de 12 études observationnelles qui ont rapporté le pourcentage des échantillons colorectaux où F. nucleatum était détecté montrent une association significativement positive entre la détection de la bactérie F. nucleatum dans le côlon et le CCR (Rapport de cotes combiné = 8,3; IC à 95 % = 5,2-13; Indice d’hétérogénéité I2 = 26,3 %; p pour hétérogénéité = 0,18). Enfin, les résultats préliminaires de l’étude de faisabilité appuient la faisabilité et l’utilité de mener une étude subséquente pour investiguer F. nucleatum dans la salive et le côlon en présence de néoplasies colorectales afin de vérifier son rôle dans l’association entre la MP et le CCR, mais des ajustements doivent être apportés à la méthodologie initiale. CONCLUSION: Nos résultats suggèrent que l’exposition à la MP peut augmenter le risque de CCR, bien qu’on ne puisse encore confirmer de lien causal. On espère que ce travail sensibilise les cliniciens, décideurs en santé et public à l’importance de prévenir et de contrôler les maladies buccodentaires, notamment la MP, pour la prévention même de maladies systémiques encore plus fatales, tels que le CCR. / BACKGROUND: Colorectal cancer (CRC) remains one of the most diagnosed cancers in Canada and around the world. Despite screening efforts, half of colorectal cancers are still diagnosed at advanced stages of the disease, thus reducing the chances of survival from this cancer. Prevention of CRC still remains essential, and efforts must be maintained to identify its modifiable risk factors. Over the past decade, there has been an accumulation of evidence on the association between colorectal enrichment in a specific bacterium, Fusobacterium nucleatum (F. nucleatum), and CRC. This has sparked debate on the association between periodontal disease (PD) and CRC, since F. nucleatum is not only a commensal bacterium of the mouth, but above all one of the main pathogens of PD. Previous epidemiological studies had indeed already suggested that PD would be associated with the risk of CRC, but their results remained inconclusive because of their methodological flaws. Dissemination of periodontal pathogen bacteria such as F. nucleatum, and of their virulence products, from the mouth to the colon and the release of chronic inflammatory mediators into the bloodstream are suggested as the mechanisms involved in the association between PD and CRC. OBJECTIVES: 1) To verify the association between PD and CRC; 2) To synthesize the literature on the association between the detection of the bacterium F. nucleatum in the colon and CRC; and 3) To verify the feasibility of investigating the concomitant expression of F. nucleatum, in both oral and colorectal environments, in the presence of colorectal neoplasms. These last two objectives are a preamble to the investigation of the role of the causative bacteria of PD, specifically F. nucleatum, as a mechanism of association between PD and CRC. METHODS: We undertook three separate research projects that address the three respective research objectives listed above: 1) A population-based case-control study (COLDENT Project) included 348 incident cases of CRC and 310 age and sex frequency-matched controls. Data were collected on PD and on major risk factors for CRC; 2) A systematic review with meta-analysis of observational studies that have investigated the bacterium F. nucleatum in colorectal samples, in CRC cases and colorectal cancerous or precancerous-free controls; and 3) A pilot hospital-based case-control study, with 22 cases of colorectal neoplasms and 21 controls, examined by colonoscopy. Saliva samples and colorectal mucosal biopsies as well as data on exposure to PD and major risk factors for CRC were collected. 8 RESULTS: The results of our population-based case-control study suggest that the rate of CRC was increased in people with a positive history of PD compared to people without a history of PD, adjusting for the majority of potential confounders (adjusted rate ratio = 1.45; 95% Confidence interval (CI): 1.04-2.01). The results of our systematic review including 24 observational studies showed that in most studies where the load of F. nucleatum in colorectal samples was quantified, it was significantly higher in CRC cases than in controls. The results of the meta-analysis of 12 observational studies that reported the percentage of colorectal samples where F. nucleatum was detected show a significantly positive association between the detection of the bacterium F. nucleatum in the colon and CRC (pooled odds ratio = 8.3; 95% CI = 5.2-13; heterogeneity index I 2 = 26.3%; p for heterogeneity = 0.18). Finally, the preliminary results of the feasibility study support the feasibility and utility of conducting a subsequent study to investigate F. nucleatum in saliva and colon in the presence of colorectal neoplasms to verify its role in the association between PD and CCR, but adjustments need to be made to the original methodology. CONCLUSION: Our results suggest that exposure to PD may increase the risk of CRC, although a causal relationship cannot yet be confirmed. It is hoped that this work will sensitize clinicians, health policy makers and the public to the importance of preventing and controlling oral diseases, including PD, for the prevention of even more fatal systemic diseases, such as CRC.