Riikka Ihalin

The sensitivity of Porphyromonas gingivalis and Fusobacterium nucleatum to different (pseudo)halide-peroxidase combinations compared with mutans streptococci

Previous studies have shown that the peroxidase system with iodide is particularly effective against Actinobacillus actinomycetemcomitans. In the present study, the effects of iodide, chloride and thiocyanate in combinations with lactoperoxidase (LP) and myeloperoxidase (MP) on the viability of Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and S. rattus were analysed. Bacteria were incubated in buffer solution containing peroxidase, substrate(s) and H2O2 (all in oral physiological concentrations), and plated after 0, 0.5 and 1 h. The oxidation product of iodide was the most bactericidal against all the bacteria tested. The effect was significantly weaker on mutans streptococci. Physiological concentrations of thiocyanate abolished the effects of LP-H2O2-iodide and MP-H2O2-iodide/chloride combinations. Thiocyanate-peroxidase systems have already been used in oral hygiene products. The incorporation of iodide into these products could make them much more potent against periodontal pathogens, and also help to prevent transmission of these pathogens from person to person via saliva.

Susceptibility of Fusobacterium nucleatum to killing by peroxidase–iodide–hydrogen peroxide combination in buffer solution and in human whole saliva

Some Gram-negative anaerobic bacteria have been associated with the infection of tooth supporting tissues, i.e. periodontitis. Of these bacteria, Fusobacterium nucleatum is sensitive to lactoperoxidase/myeloperoxidase-iodide-hydrogen peroxide system in vitro, but salivary concentrations of thiocyanate abolishes the bactericidality. These bacteria are located in periodontal pockets, on oral mucosa and in saliva. Although F. nucleatum most probably does not belong to the group of main periodontal pathogens, it sustains its proportion in the periodontal flora when gingivitis progresses to periodontitis. In this study, the sensitivity of F. nucleatum to different horseradish peroxidase-iodide-hydrogen peroxide combinations was tested both in buffer and in sterilized human whole saliva. Horseradish peroxidase was chosen because it does not bind thiocyanate at pH > or = 6. After 1h incubation at 37 degrees C, the cell viability was estimated by plate count and with flow cytometer using LIVE/DEAD BacLight kit (Molecular Probes, USA). In saliva, the horseradish peroxidase (50 microg/mL)-iodide (2.5 mM)-hydrogen peroxide (2.5 mM) combination decreased the amount of viable bacteria to 37% compared to 85% in the control without any of the components when measured with flow cytometer. Replacement of buffer by saliva decreased the bactericidality of the peroxidase system. However, in buffer less iodide and hydrogen peroxide was needed to produce significant decrease in the number of viable bacteria when measured by plate count than with flow cytometer. Our study shows that horseradish peroxidase-iodide-hydrogen peroxide combination is able to kill F. nucleatum cells in saliva. Horseradish peroxidase-iodide-hydrogen peroxide combination may be useful to diminish the degree of re-colonization of periodontitis-associated bacteria after periodontal therapy and to inhibit the transmission of these bacteria via saliva.

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.

Susceptibilities of different Actinobacillus actinomycetemcomitans strains to lactoperoxidase–iodide–hydrogen peroxide combination and different antibiotics

Actinobacillus actinomycetemcomitans has an important aetiological role in localized juvenile periodontitis and in progressive periodontitis in adults. A. actinomycetemcomitans is found mainly in periodontal pockets but also in whole saliva, a potential transmission medium. It is sensitive to peroxidase-halide systems, but the differences between periodontitis associated clinical isolates and type strains are unclear. The sensitivities of these 2 strain groups to lactoperoxidase (LP)-iodide (I(-))-hydrogen peroxide (H(2)O(2)) combinations were investigated, and the sensitivities were compared with the susceptibilities to four antibiotics. There was great variation between the sensitivities of different strains, but the 2 strain groups responded similarly. The LP (75 microg)-I(-) (100 nmol)-H(2)O(2) (1000 nmol) combination produced a similar degree of inhibition as 2 microg ampicillin. The LP-I(-) system might be a potential antimicrobial agent against A. actinomycetemcomitans transmission via saliva.

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.

Determination of safety levels of horseradish peroxidase-iodide system to human gingival keratinocytes and fibroblasts in vitro

The peroxidase-iodide (I—) system is a potential antimicrobial agent, and its bacteriocidal activity against various periodontal bacteria has been shown in many studies. The aim of this study was to investigate the possible cytotoxic effects of a non-physiological horseradish peroxidase (HRP)—I— system on human gingival keratinocytes and fibroblasts. Immortalized human skin keratinocyte cell line was used as a reference. Three indicators were studied: membrane permeability (trypan blue staining), cell growth (crystal violet staining) and metabolic activity (alamarBlue™ stain). The cells were cultured in microtitration plates, and the most commonly used exposure time to the HRP system was 1 h. The effects of HRP system on cell growth and metabolic activity were observed at lower I— and H2O2 concentrations than its effects on membrane permeability. Gingival fibroblasts were more prone to detachment than keratinocyte cell lines, but no differences in changes of growth or metabolic activities were observed between gingival fibroblasts and gingival keratinocytes. The highest concentrations of the HRP—I— system components which did not have any significant detrimental effects on the metabolic activity and cell growth of gingival keratinocytes and fibroblasts were: 50 μg/ml HRP, 500 μmol/L I— and 500 μmol/L H2O2. Although this system has been shown to be antibacterial against oral bacteria, no recommendations about the usage of the HRP—I— system in oral cavity can be made yet due to the in vitro nature of this study. Our results form the basis for future safety studies investigating the chronic toxicity of this system to oral epithelium.