Marja T. Pöllänen

Biofilm Formation Enhances the Oxygen Tolerance and Invasiveness of Fusobacterium nucleatum in an Oral Mucosa Culture Model

BACKGROUND The present study evaluates the survival capability of Fusobacterium nucleatum strains in an aerobic environment and compares the invasive capability of F. nucleatum in biofilm and planktonic forms in an organotypic cell culture (OCC) model. METHODS Biofilms of F. nucleatum American Type Culture Collection (ATCC) 25586 or Anaerobe Helsinki Negative (AHN) 9508 were produced by culturing on semipermeable membranes on brucella agar plates. The oxygen tolerance of the F. nucleatum strains was examined by incubating 3-day-old anaerobically grown biofilms in an aerobic environment (CO(2) [5% in air] incubator) for an additional 48 hours. The OCC model was constructed by seeding keratinocytes on a fibroblast-containing collagen gel. In invasion assays, a 3-day-old anaerobically grown biofilm (and planktonic bacteria in solution as the control) was placed upside down on the top of OCC and incubated under 5% CO(2) for 24 hours. Invasion of the bacteria and morphologic changes in OCC were assessed using hematoxylin and eosin, Ki-67, and periodic acid-Schiff stainings. RESULTS In biofilms, both F. nucleatum strains continuously increased their cell numbers in an aerobic environment for 48 hours. After incubating the bacterial biofilm in contact with the OCC model, F. nucleatum AHN 9508 was able to pass through the epithelial/basement membrane barrier and invade the collagen matrix. The invasiveness of biofilm F. nucleatum ATCC 25586 was limited to the epithelium. Cytotoxic effects and invasiveness of F. nucleatum on the OCC were much stronger when the bacteria were in biofilms than in the planktonic form. CONCLUSION Biofilm formation regulates the survival and invasiveness of F. nucleatum in an aerobic environment.

A Novel Organotypic Dento-Epithelial Culture Model: Effect of Fusobacterium nucleatum Biofilm on B-Defensin-2,-3, and LL-37 Expression

BACKGROUND In the present study, the expression and localization of three epithelial peptides (human β-defensin [hBD]-2 and -3, and cathelicidin [LL-37]) are studied in an organotypic dento-epithelial (OD-E) model exposed to Fusobacterium nucleatum (Fn) biofilm. METHODS Biofilm of Fn ATCC 25586 or AHN 9508 were produced by culturing each strain on semipermeable membranes. The OD-E model was constructed by seeding keratinocytes on fibroblast-containing collagen gels and by placing dentin pieces on the top. A 3-day-old biofilm was placed on the top of the OD-E and the coculture was incubated for 5 hours or 24 hours. Production of epithelial antimicrobial peptides was determined immunohistochemically. RESULTS After 5 hours of incubation, the biofilm of each Fn strain stimulated expression of hBD-2 and -3. hBD-2 was localized on superficial layers and hBD-3 on basal cell layers of the epithelium and dento-epithelial junctions, whereas LL-37 was only weakly expressed. After 24 hours, hBD-2 expression was extended toward basal cell layers of the epithelium. In contrast, hBD-3 expression extended toward superficial layers of the epithelium. In the case of Fn AHN 9508 biofilm, LL-37 was localized in the cell layers of the dento-epithelial junction. CONCLUSION In our OD-E model, epithelial antimicrobial peptide responses to Fn biofilms have distinct regulation and localization characteristics, resembling those known to occur in the gingival epithelium in vivo.

Environmental Stimuli Shape Biofilm Formation and the Virulence of Periodontal Pathogens

Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.

Identification of a novel bacterial outer membrane interleukin-1Β-binding protein from Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative opportunistic oral pathogen. It is frequently associated with subgingival biofilms of both chronic and aggressive periodontitis, and the diseased sites of the periodontium exhibit increased levels of the proinflammatory mediator interleukin (IL)-1β. Some bacterial species can alter their physiological properties as a result of sensing IL-1β. We have recently shown that this cytokine localizes to the cytoplasm of A. actinomycetemcomitans in co-cultures with organotypic gingival mucosa. However, current knowledge about the mechanism underlying bacterial IL-1β sensing is still limited. In this study, we characterized the interaction of A. actinomycetemcomitans total membrane protein with IL-1β through electrophoretic mobility shift assays. The interacting protein, which we have designated bacterial interleukin receptor I (BilRI), was identified through mass spectrometry and was found to be Pasteurellaceae specific. Based on the results obtained using protein function prediction tools, this protein localizes to the outer membrane and contains a typical lipoprotein signal sequence. All six tested biofilm cultures of clinical A. actinomycetemcomitans strains expressed the protein according to phage display-derived antibody detection. Moreover, proteinase K treatment of whole A. actinomycetemcomitans cells eliminated BilRI forms that were outer membrane specific, as determined through immunoblotting. The protein was overexpressed in Escherichia coli in both the outer membrane-associated form and a soluble cytoplasmic form. When assessed using flow cytometry, the BilRI-overexpressing E. coli cells were observed to bind 2.5 times more biotinylated-IL-1β than the control cells, as detected with avidin-FITC. Overexpression of BilRI did not cause binding of a biotinylated negative control protein. In a microplate assay, soluble BilRI bound to IL-1β, but this binding was not specific, as a control protein for IL-1β also interacted with BilRI. Our findings suggest that A. actinomycetemcomitans expresses an IL-1β-binding surface-exposed lipoprotein that may be part of the bacterial IL-1β-sensing system.

Trimeric form of intracellular ATP synthase subunit β of Aggregatibacter actinomycetemcomitans binds human interleukin-1β.

Bacterial biofilms resist host defenses and antibiotics partly because of their decreased metabolism. Some bacteria use proinflammatory cytokines, such as interleukin (IL)-1β, as cues to promote biofilm formation and to alter virulence. Although one potential bacterial IL-1β receptor has been identified, current knowledge of the bacterial IL-1β sensing mechanism is limited. In chronic biofilm infection, periodontitis, Aggregatibacter actinomycetemcomitans requires tight adherence (tad)-locus to form biofilms, and tissue destroying active lesions contain more IL-1β than inactive ones. The effect of IL-1β on the metabolic activity of A. actinomycetemcomitans biofilm was tested using alamarBlue™. The binding of IL-1β to A. actinomycetemcomitans cells was investigated using transmission electron microscopy and flow cytometry. To identify the proteins which interacted with IL-1β, different protein fractions from A. actinomycetemcomitans were run in native-PAGE and blotted using biotinylated IL-1β and avidin-HRP, and identified using mass spectroscopy. We show that although IL-1β slightly increases the biofilm formation of A. actinomycetemcomitans, it reduces the metabolic activity of the biofilm. A similar reduction was observed with all tad-locus mutants except the secretin mutant, although all tested mutant strains as well as wild type strains bound IL-1β. Our results suggest that IL-1β might be transported into the A. actinomycetemcomitans cells, and the trimeric form of intracellular ATP synthase subunit β interacted with IL-1β, possibly explaining the decreased metabolic activity. Because ATP synthase is highly conserved, it might universally enhance biofilm resistance to host defense by binding IL-1β during inflammation.

Host-bacteria crosstalk at the dentogingival junction.

The dentogingival junction is of crucial importance in periodontal host defense both structurally and functionally. Oral bacteria exert a constant challenge to the host cells and tissues at the dentogingival junction. The host response is set up to eliminate the pathogens by the innate and adaptive defense mechanisms. In health, the commensal bacteria and the host defense mechanisms are in a dynamic steady state. During periodontal disease progression, the dental bacterial plaque, junctional epithelium (JE), inflammatory cells, connective tissue, and bone all go through a series of changes. The tissue homeostasis is turned into tissue destruction and progression of periodontitis. The classical study of Slots showed that in the bacterial plaque, the most remarkable change is the shift from gram-positive aerobic and facultatively anaerobic flora to a predominantly gram-negative and anaerobic flora. This has been later confirmed by several other studies. Furthermore, not only the shift of the bacterial flora to a more pathogenic one, but also bacterial growth as a biofilm on the tooth surface, allows the bacteria to communicate with each other and exert their virulence aimed at favoring their growth. This paper focuses on host-bacteria crosstalk at the dentogingival junction and the models studying it in vitro.

Interleukin-1β is internalised by viable Aggregatibacter actinomycetemcomitans biofilm and locates to the outer edges of nucleoids

The opportunistic pathogen Aggregatibacter actinomycetemcomitans causes periodontitis, which is a biofilm infection that destroys tooth-supportive tissues. Interleukin (IL)-1β, a central proinflammatory cytokine of periodontitis, is an essential first line cytokine for local inflammation that modulates the cell proliferation and anti-pathogen response of human gingival keratinocytes. Previously, we demonstrated that A. actinomycetemcomitans biofilms bind IL-1β; however, whether this binding is an active process is not known. In this study, we showed for the first time with immuno-electron microscopy that viable bacterial biofilm cells internalised IL-1β when co-cultured with an organotypic mucosa. Decreased biofilm viability hindered the ability of biofilm to sequester IL-1β and caused IL-1β leakage into the culture medium. In some A. actinomycetemcomitans cells, intracellular IL-1β localized to the outer edges of the nucleoids. We identified the DNA-binding protein HU as an IL-1β interacting protein with mass spectroscopy and showed the interaction of recombinant HU and IL-1βin vitro using enzyme-linked immunosorbent assay (ELISA). Close contact with a viable A. actinomycetemcomitans biofilm decreased the proliferation and apoptosis of human gingival keratinocytes as demonstrated using Ki-67 and the terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, respectively. Our results suggest that viable A. actinomycetemcomitans biofilms may disturb the critical first steps of local inflammation in periodontitis by binding and internalising IL-1β. The interaction of IL-1β with conserved HU provides a potential mechanism for shaping bacterial gene expression.

A novel intrinsically disordered outer membrane lipoprotein of Aggregatibacter actinomycetemcomitans binds various cytokines and plays a role in biofilm response to interleukin-1β and interleukin-8

ABSTRACT Intrinsically disordered proteins (IDPs) do not have a well-defined and stable 3-dimensional fold. Some IDPs can function as either transient or permanent binders of other proteins and may interact with an array of ligands by adopting different conformations. A novel outer membrane lipoprotein, bacterial interleukin receptor I (BilRI) of the opportunistic oral pathogen Aggregatibacter actinomycetemcomitans binds a key gatekeeper proinflammatory cytokine interleukin (IL)-1β. Because the amino acid sequence of the novel lipoprotein resembles that of fibrinogen binder A of Haemophilus ducreyi, BilRI could have the potential to bind other proteins, such as host matrix proteins. However, from the tested host matrix proteins, BilRI interacted with neither collagen nor fibrinogen. Instead, the recombinant non-lipidated BilRI, which was intrinsically disordered, bound various pro/anti-inflammatory cytokines, such as IL-8, tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Moreover, BilRI played a role in the in vitro sensing of IL-1β and IL-8 because low concentrations of cytokines did not decrease the amount of extracellular DNA in the matrix of bilRI− mutant biofilm as they did in the matrix of wild-type biofilm when the biofilms were exposed to recombinant cytokines for 22 hours. BilRI played a role in the internalization of IL-1β in the gingival model system but did not affect either IL-8 or IL-6 uptake. However, bilRI deletion did not entirely prevent IL-1β internalization, and the binding of cytokines to BilRI was relatively weak. Thus, BilRI might sequester cytokines on the surface of A. actinomycetemcomitans to facilitate the internalization process in low local cytokine concentrations.

Interactions between the Aggregatibacter actinomycetemcomitans secretin HofQ and host cytokines indicate a link between natural competence and interleukin-8 uptake

ABSTRACT Naturally competent bacteria acquire DNA from their surroundings to survive in nutrient-poor environments and incorporate DNA into their genomes as new genes for improved survival. The secretin HofQ from the oral pathogen Aggregatibacter actinomycetemcomitans has been associated with DNA uptake. Cytokine sequestering is a potential virulence mechanism in various bacteria and may modulate both host defense and bacterial physiology. The objective of this study was to elucidate a possible connection between natural competence and cytokine uptake in A. actinomycetemcomitans. The extramembranous domain of HofQ (emHofQ) was shown to interact with various cytokines, of which IL-8 exhibited the strongest interaction. The dissociation constant between emHofQ and IL-8 was 43 nM in static settings and 2.4 μM in dynamic settings. The moderate binding affinity is consistent with the hypothesis that emHofQ recognizes cytokines before transporting them into the cells. The interaction site was identified via crosslinking and mutational analysis. By structural comparison, relateda type I KH domain with a similar interaction site was detected in the Neisseria meningitidis secretin PilQ, which has been shown to participate in IL-8 uptake. Deletion of hofQ from the A. actinomycetemcomitans genome decreased the overall biofilm formation of this organism, abolished the response to cytokines, i.e., decreased eDNA levels in the presence of cytokines, and increased the susceptibility of the biofilm to tested β-lactams. Moreover, we showed that recombinant IL-8 interacted with DNA. These results can be used in further studies on the specific role of cytokine uptake in bacterial virulence without interfering with natural-competence-related DNA uptake.

Behavioural interventions that have the potential to improve self-care in adults with periodontitis: a systematic review

Abstract Aim: To evaluate behavioural and educational interventions used to improve self-care in adult periodontitis patients in comparison with conventional instruction. Methods: A systematic electronic search of empirical studies that were published up to June 2017 using the MEDLINE database was performed. The reference lists of all of the included studies and articles from six separate journals were manually searched. Results: A total of 1806 articles were identified. Six articles fulfilled the inclusion and exclusion criteria. The interventions used in periodontal treatment had theoretical backgrounds of cognitive behavioural approach, self-regulation theory of Leventhal, motivational interviewing and a client self-care commitment model. The control group in each study was described receiving conventional information. The outcomes of the interventions were classified into three categories: 1) clinical findings 2) self-reported self-care and 3) patient evaluations of the intervention. The behavioural intervention groups seemed to perform slightly better than the control groups when clinical outcome measures such as the presence of plaque or number of periodontal pockets were used. Furthermore, behavioural interventions increased patient reported compliance (e.g. effectiveness of self-care and frequency of interdental cleaning). The different behavioural techniques all seemed to work more effectively than conventional instruction. No behavioural technique could be identified superior to the other. Conclusions: The behavioural interventions seem to be beneficial for patient adherence and may therefore improve periodontal treatment success. However, there is a need to further explore the use of different methods in studies with larger sample sizes, longer follow-up times and both behavioural and clinical outcome measures.