Mechanistic insight into the membrane disrupting properties of thymol in Candida species

Abstract

Candida infection is a major problem in immunocompromised patients and has become a significant public health concern in recent years due to high toxicity, multidrug resistance and unfavourable side effects of conventional antifungal drugs. This study aims to investigate the antifungal efficacy of thymol, a natural monoterpenoid, on the membrane integrity of Candida albicans, Candida glabrata and Candida tropicalis. Minimum inhibitory concentrations (MICs) of thymol were 32 µg/ml, 63 µg/ml, and 125 µg/ml for C. albicans, C. tropicalis and C. glabrata, respectively. The minimum fungicidal concentrations (MFC) were 64 µg/ml, 110 µg/ml and 240 µg/ml, respectively. At MIC, thymol treated cells showed complete suppression of cell growth (growth kinetics, disc diffusion), altered morphology (scanning electron microscopy), leakage of intracellular macromolecules (absorption at 260 nm), significant reduction in ergosterol levels and inhibition of plasma membrane H+-ATPase (Pma1) activity. The disruption of the fungal membranes by thymol was further confirmed by propidium iodide uptake using flow cytometry and confocal microscopy. In silico studies with membrane-bound proteins (lanosterol 14-α demethylase (LDM) and Pma1), showed good binding affinity of −6.3 kcal/mol and −6.7 kcal/mol, respectively, and formed hydrogen bonds with crucial amino acid residues at the active site of target proteins. With low MIC values, negligible host toxicity, desirable ADME properties, thymol has immense potential as a potent antifungal drug that can be used for developing mechanism-based drugs as it has specific binding affinity for specific target proteins. Studies using animal models are required for further validation.