Limonene inhibits virulence associated traits in Candida albicans: In-vitro and in-silico studies

Abstract

Background: Multidrug resistance and undesirable side effects of currently available antifungal drugs, has made treatment of Candidiasis difficult in immunocompromised patients. Limonene, commonly found in citrus essential oils, has immense therapeutic potential. Purpose: Efficacy of this monoterpene was studied against virulence attributes of C. albicans. Methods: Percentage haemolysis, antifungal susceptibility, time kill studies, secretion of hydrolytic enzymes, morphological transition, adhesion and biofilm formation (XTT reduction assay, scanning electron microscopic and calcofluor white stained fluorescence imaging) were studied in the presence of limonene. Molecular docking by InstaDock software followed by MD simulation studies (AMBER software suite) assessed the limonene-protein interactions with five virulence associated antifungal targets (Als3, Bcr1, Plb1, Sap2 and Tec1). Results: With MIC and MFC values of 300 µg/mL and 400 µg/mL, respectively, it caused only 1% haemolysis (3 times less than fluconazole) and significantly reduced adhesion to buccal epithelial cells. An incubation of 9 h at 2MIC reduced growth by 100%. At MIC, secretion of proteinases and phospholipases was reduced by 73% and 53%, respectively. Adhesion and biofilm formation was reduced by 91% and 87%, respectively. Biofilm biomass formed on silicon sheets was inhibited by 69%. Limonene treatment disrupted biofilm structure and integrity. Filamentation, studied in four different hyphae inducing media, was significantly reduced. Although stable hydrophobic interactions were observed with all target proteins, limonene gave a good docking score with Plb1 (−6.0 kcal/mol) and Tec1 (−5.7 kcal/mol). Complexes formed with both these proteins showed stable RMSD profiles except with Bcr1. Conclusion: Limonene binds to proteins critical to Candida pathogenicity and inhibits adhesion, biofilm formation and morphological transition. It is nontoxic and has immense potential in the management of invasive candidiasis.