Currently, the treatment options for fungal infections, such as oral candidosis, present limitations due to the low availability of antifungal drugs and the emergence of drug resistant strains. The use of probiotics is considered promising because they have antimicrobial activity as well as immunomodulatory action, but its clinical application in immunocompromised patients is critical due to the possibility of bacteraemia caused by bacteria of the genus Lactobacillus. In this context, the isolation of the bioactive substances produced by Lactobacillus for the control of oral candidiasis becomes innovative and safe for hospitalized and immunocompromised patients. Therefore, the objective of this study will be to purify and identify the metabolites produced by L. paracasei 28.4 and to evaluate their in vitro effects on C. albicans virulence and its antimicrobial and immunological response in in vivo studies models. The metabolites of L. paracasei 28.4 culture will be extracted with ethyl acetate and subsequently fractionated and identified by High-performance liquid chromatography with diode array detection (CLAE-DAD). Initially, the crude extract and all fractions obtained from the supernatant will be evaluated for cytotoxicity in cell culture of human gingival fibroblasts (FMM-1), mouse macrophages (RAW 264.7) and human skin keratinocytes (HaCaT). Next, the crude extract and supernatant fractions of L. paracasei 28.4 will be tested on 30 clinical strains of C. albicans isolated from the oral cavity of patients with Human Immunodeficiency Virus (HIV) using the Minimum Inhibitory Concentration assay (MIC). The antifungal effect of the crude extract and supernatant fractions of L. paracasei 28.4 will be verified in two C. albicans strains fluconazole resistant using in vitro assays of biofilm formation and filamentation. Also in vitro, the immunomodulatory action of the crude extract and supernatant fractions of L. paracasei 28.4 will be investigated using Candida-activated RAW 264.7 cells for quantification of cytokines using fluorescently labeled beads. Next, the prophylactic and therapeutic effects of the crude extract and fractions of L. paracasei 28.4 supernatant will be investigated in the experimental model of Galleria mellonella by analysis of survival curve, hemocyte count, hemocyte cell characterization by flow cytometry, C. albicans counts of the fatty body and genes expression related to antimicrobial peptides (galiomycin, galleriomycin, cepropin D), phagocytosis (C8-Contig 1910) and the regulation of cytokines and cascade NF-kB (C7 Contig 15362) present on the immune response of this invertebrate model. Therefore, the fraction of L. paracasei 28.4 supernatant with better antifungal and immunomodulatory effect observed in the previous assays will be selected with the purpose of expanding and confirming such effects in the oral candidiasis in immunosuppressed mice. This bioactive substance will be applied on lesions induced by candidiasis in the tongue of animals. The development of oral candidiasis will be monitored recoverying of C. albicans from the oral cavity of the animals, macroscopic evaluation, histological analysis and gene expression of beta defensins 1 and 3. The results will be submitted to an exploratory analysis for selection of the most appropriate statistical test for each experiment in this study. The level of significance considered for each test will be 5%.
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