Fig 1.
Functional classification and abundance levels of transcripts regulated in Paracoccidioides lutzii in the presence of argentilactone obtained by RNAseq.
(A) Total transcripts represented by classified and unclassified categories; (B) Genes differentially expressed in the presence of argentilactone; (C) Down-regulated genes in the presence of argentilactone; (D) Up-regulated genes in the presence of argentilactone. Counting the number of reads for each gene in the genome annotated reference was applied to a statistical analysis using a Fisher's exact test for the identification of genes with differential expression.
Fig 2.
Ethanol dosage in Paracoccidioides lutzii under the action of argentilactone.
A total of 106 cells were used for each sample, and the ethanol levels in the presence and absence of argentilactone were quantified using enzymatic detection. The data are expressed as the mean ± standard deviation of biological triplicates of independent experiments. Student's t-test was used. *, significantly different from the carbon condition at a p-value of ≤ 0.05.
Fig 3.
Effect of argentilactone on the polymer levels of the Paracoccidioides lutzii cell wall.
Fluorescence microscopy showing yeast cells stained by CFW and CR: (A) P. lutzii control stained with CR; (B) P. lutzii after treatment with argentilactone, stained with CR; (C) P. lutzii control stained with CFW; (D) P. lutzii after treatment argentilactone, stained with CFW.
Fig 4.
Effect of argentilactone on transcript expression levels in Paracoccidioides lutzii yeast cells.
The expression levels of hsp90, ccp, sod and rbt5 genes in Paracoccidoides spp. yeast cells grown in MMcM liquid medium with or without argentilactone were analyzed. The data were normalized using the constitutive gene encoding α-tubulin as the endogenous control and are presented as relative expression in comparison to the experimental control cells, whose value was set to 1. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. *, significantly different from the control at a p-value of ˂ 0.05.
Fig 5.
Susceptibility of Paracoccidioides brasiliensis yeast cells exposed to argentilactone.
Samples containing 106, 105 and 104 Pbsod-aRNA, Pbhsp90-aRNA (A), Pbccp-aRNA and Pbrbt5-aRNA (B) yeast cells were spotted in solid Fava Netto’s supplemented with argentilactone at the concentrations of 4.5, 9, 18 and 36 μg/mL. Control cells, wild type (WT) and empty vector (EV) were assayed without argentilactone. The plates were incubated for 7 days at 36°C before photo documentation.
Fig 6.
Superoxide dismutase activity.
Yeast cells were grown in the presence of argentilactone for 6 h, and total proteins were extracted and used to measure superoxide dismutase activity. Student’s t test was used for statistical comparisons, and the observed differences were statistically significant (p ˂ 0.05). The error bars represent the standard deviation of three biological replicates.
Fig 7.
Effect of argentilactone on the mitochondrial membrane potential of Paracoccidioides lutzii.
The mitochondrial membrane potential was determined by flow cytometry analysis of yeast cells treated with antimycin A (A) or argentilactone (B) for 6 h and stained with rhodamine 123. Control cells without treatment (C) were used in the test.