Investigação sobre o mecanismo da ação antifúngica de uma osmotina de Calotropis procera contra cepas de Candida

Abstract

Osmotins are defense proteins (PR-5) that exhibit antifungal activity. An osmotin isolated from Calotropis procera (CpOsm) has shown activity against phytopathogenic fungi; however, its effects against Candida, a medically important yeast genus, remain poorly explored. To address this gap, this study aimed to investigate the antifungal activity and mechanism of action of CpOsm against strains of three Candida species (C. albicans, C. tropicalis, and C. auris). For this purpose, the protein was produced in Escherichia coli BL21(DE3) and purified by affinity chromatography. The recombinant osmotin (rCpOsm) exhibited antifungal activity against all strains tested, including fluconazole-resistant isolates. Minimum inhibitory concentration (MIC) values were 1.49 nM for C. albicans, 0.373 nM for C. tropicalis, and 2.99 nM for C. auris, whereas minimum fungicidal concentration (MFC) values were 2.99 nM, 1.49 nM, and 5.97 nM, respectively. rCpOsm demonstrated significant anti-biofilm activity (p < 0.05) against fluconazole-sensible and fluconazole-resistant C. auris strains, with a dual effect: inhibition of biofilm formation and disruption of mature biofilms. At 10×MIC concentration, the protein significantly reduced biomass and metabolic activity by up to 92% and 97% (biofilms in formation) and 57.5% and 86.0% (mature biofilms), as assessed by crystal violet and MTT assays. Investigation of the mechanism of action in vitro suggested that the antifungal effect on C. aurisis mediated by an interaction of rCpOsm with ergosterol in the yeast plasma membrane, as evidenced by an increase in MIC (> 10-fold) in the presence of exogenous ergosterol. Fluorescence microscopy assays revealed that CpOsm compromised membrane integrity and increased the production of reactive oxygen species in both planktonic cells and biofilms of C. auris. These cellular alterations were accompanied by changes in the protein profile of C. auris cells treated with CpOsm. Proteomic analysis revealed significant alterations in 512 proteins of C. auris, of which 505 showed an increase in abundance [Log2(fold change) > 1.5], mainly related to lipid metabolism, oxidative stress response, cell wall remodeling, and ionic homeostasis, while structural and proliferative proteins showed reduced abundance [Log2(fold change) < 0.5] or were no longer detected. Maintenance of fungicidal activity at low concentrations indicates that adaptive mechanisms are insufficient to restore cellular viability. Collectively, these findings highlight rCpOsm as a promising molecule for the development of novel antifungal strategies against clinically relevant drug-resistant pathogens.