Project description:Previous work has reported that the peptide derived from LfcinB, R-1-R exhibited anti-Candida activity, which is enhanced when combined with an extract from the Bidens pilosa plant. However, the mechanism of action has not been studied until now. In the current study, an approach to the mechanism of action was carried out through the use of mutant strains of C. albicans, a proteomic approach, followed by a bioinformatic analysis, and a validation of the results obtained, through biological assays in both SC5314 strain and a fluconazole-resistant isolate of Candida albicans, after incubation with the treatments. The results obtained with the mutant strains suggest that the R-1-R peptide and the B. pilosa extract exhibit a mechanism of action related to cell wall stress mediated by MAPKs (Hog1p and Mkc1p) and alteration in mitochondrial respiration due to interruption of ETC. After, changes in the protein expression profile of FLC-sensitive and -resistant C. albicans after six h of incubation with R-1-R or combination were analyzed, using a label free quantitative proteomic approach. The proteomic data showed that after treatment with R-1-R in the two study strains, most of the differentially expressed proteins in comparison to controls were up-regulated. These proteins were mainly involved in membrane and cell wall biosynthesis, membrane transporters, oxidative stress, the mitochondrial respiratory chain, and response to DNA damage. Besides, the proteomic analysis of C. albicans parental strain SC5314 treated with R-1-R in combination with an ethanolic extract of B. pilosa was also undertaken. The proteins differentially expressed after this treatment were involved in similar functional processes as the proteins differentially expressed after treatment with R-1-R peptide alone, but at different levels and were mostly down-regulated. The biological assays allowed validation of the proteomic results, evidencing damage to the cell surface, generation of reactive oxygen species and decrease in mitochondrial membrane potential. The results provide information to begin to unlock the understanding of the complex antifungal mode of action of the R-1-R peptide, its combination with the extract, and possibly other derivatives of natural products.
Project description:Bidens pilosa (BP) is an edible Asteraceae plant found worldwide that has traditionally been used as food without noticeable side effects. BP has also been used as an herbal medicine to treat over 41 categories of disease in humans and animals. However, to date no long-term toxicity study of BP has been conducted in animals. In this study, 24-week oral toxicity of BP at doses of 0%, 0.5%, 2.5%, 5% and 10% of food was investigated in mice. Mortality, body weight, organ weight, food intake, water consumption, hematology, serum biochemistry, urinalysis, genotoxicity and organ histopathology of animals of both sexes were analyzed. No significant difference in the above parameters was observed between control and BP-fed mice except that body weight and food intake in those fed with 10% BP were significantly less than controls. In addition, similar results were seen in chickens fed with BP for 28 days. Collectively, the data demonstrate that BP has no adverse effects in mice and chickens at dose of 5% or less of food.