Object
Title: Modified lactoperoxidase system as a limiting factor in the development of cariogenic Streptococcus mutans biofilm
Abstract:
Introduction The lactoperoxidase system is non-specific, non-cellular part of the immune system that is active in biological fluids such as saliva, tears, respiratory tract fluid, milk and female reproductive track fluid. This enzymatic system plays an important role in mucosal antimicrobial defence. Lactoperoxidase system consists of lactoperoxidase (LPO) – a haemoprotein enzyme, LPO substrates (hydrogen peroxide and (pseudo)halide) and products of enzymatic oxidation of pseudohalides formed in (pseudo)halogenation cycle. The main substrate of the LPO system in vivo is thiocyanate (SCN-), which is oxidised to hypothiocyanite (HOSCN/OSCN-). The anitimicrobial activity of hypothicyanite is due to its ability to oxidize sulfhydryl groups in microbial proteins, resulting in the loss of their biological function. Although, in the published studies, the LPO system shows antimicrobial activity against microorganisms in their planctonic form, the antibiofilm or antimicrobial activity against microorganisms within a biofilm structure is drastically weakened or even statistically insignificant. It should be noted that lactoperoxidase is capable of oxidizing other (pseudo)halides, which are physiologically absent or present in insignificant concentrations in biological fluids, to products with much greater oxidative potential than the physiological product. Examples of alternative (pseud ; o)halide substrates include iodide, which is oxidized to several different reactive iodide species (depending on environmental conditions), selenocyanate, which is oxidized to hyposelenocyanite, and finally the mixture of thiocyanate and iodide, which as a result of enzymatic reaction and subsequent reactions is oxidized to highly toxic cyanogen iodide. All of the above mentioned products, due to their higher reactivity, may also be characterized by stronger antibiofilm effect. Similar to other peroxidases, lactoperoxidase has the ability to oxidize organic compounds in the peroxidation cycle. Substrates include hydrogen peroxide and a large number of organic compounds (simple phenolic compounds including polyphenols, peptides, proteins, drugs). With regard to antimicrobial defense, entering the peroxidation cycle is undesirable. First, it consumes hydrogen peroxide ( whose supply in vivo is limited) for the purpose of peroxidation cycle without antimicrobial effectiveness. Secondly, the peroxidation cycle consists of two successive reactions in which an organic molecule is oxidized and as a result of the first reaction the enzyme is transformed into Compound II, which is a highly stable and rather unreactive form of lactoperoxidase. Compound II reacts relatively slowly with some organic substrates and its accumulation leads to the depletion of enzyme potentially capable of syn ; thesizing antimicrobial products. The accumulation of Compound II occurs in the situation of increased availability of hydrogen peroxide during the inflammatory process, which consequently causes a decrease in thr antimicrobial action of the lactoperoxidase system when it is most needed. Aim of the studies The aim of the studies was to attempt to answer the question of whether there is a possibility to enhance the antibiofilm action of the lactoperoxidase system by modifying it by 1) using nonphysiological (pseudo)halide substrates and 2) increasing the rate of synthesis of antimicrobial products using reactivators (derived from the rhizome of Reynoutria japonica, R. sachalinensis and R. x bohemica) of the highly stable and unreactive Compound II. Methods The following methods were used to answer the above question . High purity bovine milk lactoperoxidase was used in all experiments. In the studies focused on the assessment of the possibility of activation of the lactoperoxidase system to increase the rate of synthesis of antimicrobial product, the following methods were applied 1) kinetic spectrophotometric assay of the rate oxidation of Ellman’s reagent to assess the reactivating effect of the extracts and their fractions from rhizome of three species of genus Reynoutria and four polyphenolic compounds representative of main polyphenolic groups identified in the plant materi ; al; 2) stopped-flow UV/VIS spectroscopy to evaluate the activating/inhibitory effect of the studied extracts/fractions/compounds; 3) kinetic monitoring of the optical density over time of Streptococcus mutans culture in the presence of lactoperoxidase system and individual extracts/fractions/polyphenols; 4) MIC and MBC assay for individual substances in order to exclude their own animicrobial effect, which consequently confirmed their activating effect on the lactoperoxidase system. The evaluation of the antibiofilm effect of the lactoperoxidase system, modified by the use of nonphysiological (pseudo)halide substrates, was based on the cariogenic biofilm model of Streptococcus mutans, cultured in polistyrene plates in a sucrose-rich environment. The following methods were used to evaluate the antibiofilm effect 5) total biofilm biomass assay with crystal violet; 6) insoluble polysaccharide mass assay with anthrone; 7) bioamperometric kinetic assay of lactate, glucose and sucrose concentration in biofilm growth media; 8) activity assay of phosphotransferase system responsible for intracellular cabrohydrate transfer; 9) biofilm NAD+ and NADH concentration assay using capillary electrophoresis. The in vitro toxicity of the investigated variations of the lacotperoxidase system was evaluated in an adherent cellular culture model of human gingival fibroblasts. The following technique ; s were applied in the study 10) evaluation of proapoptotic effect using flow cytometry with annexin V conjugated with fluorescent dye; 11) cell cycle assay using flow cytometry combined with propidium iodide staining after intracellular RNA digestion; 12) intracellular glutathione reserve assay using flow cytometry and glutathione binding fluorescent probe; 13) superoxide production in the cells assessed by flow cytometry and MitoSOX staining. The results The conducted studies on the effect of Reynoutria sp. extracts and their fractions as well as pure polyphenols have proved the following. 1) Polyphenolic compounds can be both activators and inhibitors of the (pseudo)halogenation cycle of lactoperoxidase. 2) The strongest activating effect was obtained with dichloromethane and the diethyl ether fraction from R. x bohemica, and among the pure polyphenols, (-)-epicatechin showed an activating effect. 3) The observed activation was related to the rapid reaction of polyphenols with Compound II, converting it to the native LPO form. 4) Polyphenol-rich extract fractions containing compounds with high molecular weight and high degree of cross-linking, as well as the representative of such compounds – vanicoside B showed inhibitory effect on (pseudo)halogenation cycle of lactoperoxidase. 5) It was confirmed, that the phenomenon of activation of the lactoperoxidase system increases its ; antimicrobial activity against S. mutans. The analysis of the effect of the modified lactoperoxidase system using different substrates has demonstrated that 6) the physiological lactoperoxidase system with thiocyanate as (pseudo)halide substrate does not exhibit any significant antibiofilm effect or anticariogenic effect against S. mutans biofilm. 7) In contrast, the system in which thiocyanate was replaced by iodide had the strongest antibiofilm and anticariogenic properties among the modifications tested, decreasing the total biofilm biomass and reducing the mass of unsoluble polisaccharide, impairing intracellular carbohydrate transport, inhibiting lactic acid synthesis and reducing the overall redox transformations manifested by a decrease in intracellular NAD+ and NADH concentrations. 8) The system that synthesized cyanogen iodide, contrary to expectations, did not demonstrate significant antibiofilm properties. 9) The system containing selenocyanate as substrate showed a moderate antibiofilm effect. In vitro toxicity studies of lactoperoxidase system modified with alternative substrates showed that 11) each of the evaluated systems, including the physiological lactoperoxidase system, revealed statistically significant toxic effect. 12) This effect was a result of an increase in cell membrane permeability of the cells incubated with the tested systems. 13) The toxic effec ; t was not found to be related to an increase in oxidative stress. 14) The strongest toxic effect was observed when the iodide was the (psedo)halide substrate. Conclusions The presented research suggests that it is possible to modify the lactoperoxidase system in such a way that it may exhibit antimicrobial properties not only against planktonic microorganisms but also against biofilms. It has been clearly pointed out that lactoperoxidase has the potential as a biomolecule useful in combating pathogenic biofilms especially those related to the formation of dental caries.
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Rada Dyscypliny Nauki medyczne
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Feb 17, 2025
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Feb 17, 2025
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http://dl.cm-uj.krakow.pl:8080/publication/5201
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| Edition name | Date |
|---|---|
| UJCMa7232a9b5028414fb24ec6ae0d64634c | Feb 17, 2025 |
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