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Antibiofilm activity of lichen secondary metabolites

Abstract : The oral bacteria do not only infect the mouth and reside there, but also travel via the blood and reach distant body organs. If left untreated, the dental biofilm that can cause destructive inflammation in the oral cavity may result in serious systemic medical complications. In dental biofilm, Streptococcus gordonii, a primary oral colonizer, constitutes the platform on which late pathogenic colonizers like Porphyromonas gingivalis, the causative agent of periodontal diseases, will bind. The aim of the first study was to determine the antibacterial activity of eleven natural lichen compounds belonging to different chemical families to uncover new antibiotics which can fight against the oral bacteria. Three compounds were shown to have promising antibacterial activities where psoromic acid had the lowest MICs of 11.72 and 5.86 µg/mL against S. gordonii and P. gingivalis, respectively. Novel butyrolactone analogues were then designed and synthesized based on the known lichen antibacterial compounds, lichesterinic acids (B-10 and B-11), by substituting different functional groups on the butyrolactone ring trying to enhance its activity on S. gordonii and P. gingivalis.. Among the derivatives, B-12 and B-13 had the lowest MIC of 9.38 µg/mL where they have shown to be stronger bactericidals, by 2-3 times, than the reference antibiotic, doxycycline. B-12 and B-13 were also the most efficient on P. gingivalis exhibiting MIC of 0.037 and 0.293 µg/mL and MBC of 1.17 and 0.586 µg/mL, respectively. These 2 compounds were then checked for their cytotoxicity against human gingival epithelial cells and macrophages by MTT and LDH assays which confirmed their safety against the tested cell lines. A preliminary study of the structure-activity relationships unveiled the important dual role contributed by two substituents, alkyl chain at C4 and carboxyl group at C5 positions, in their mechanism of action. This was followed by the investigation of B-12 and B-13 for their antibiofilm activity against both oral strains using crystal violet assay and confocal microscopy. Both derivatives displayed a lowest concentration with maximal biofilm inhibition, LCMI, of 9.38 µg/mL against S. gordonii and 1.17 µg/mL against P. gingivalis. However, when sub-inhibitory concentrations of B-12 and B-13 were used, we demonstrated that the two investigated strains were able to form biofilms in vitro. Indeed, this antibiofilm activity decreased as indicated by the expression of the genes implicated in adhesion and biofilm formation. To better understand the mechanism of action of butyrolactones, we have investigated B-13 bacterial localization by synthesizing a fluorescently labeled B-13 with NBD (4-nitro-benzo[1,2,5]oxadiazole) conserving its antibacterial activity. By confocal microscope, we showed that this compound binds to S. gordonii cell surface and this was also demonstrated by HPLC analysis. By adhering to cell surface, B-13 induced cell wall disruption leading to the release of bacterial constituents and consequently, the death of S. gordonii, a Gram-positive bacterium. The expression of two genes, murA and alr, implicated in cell wall synthesis, was modified in the presence of this butyrolactone. Gram-negative bacteria such as P. gingivalis showed also cracked and ruptured cells in the presence of B-13, suggesting that this butyrolactone acts on Gram-positive and Gram-negative strains, but with greater efficacy against the Gram-negatives. Besides, we also demonstrated that the analogue of B-13, B-12, has also induced disruption of P. gingivalis and S. gordonii. All these studies demonstrated that butyrolactones derived from a lichen metabolite can be proposed as potent antibacterial agents against oral pathogens causing serious medical complications.
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Alaa Sweidan. Antibiofilm activity of lichen secondary metabolites. Human health and pathology. Université Rennes 1, 2017. English. ⟨NNT : 2017REN1B017⟩. ⟨tel-01895602⟩

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