Functional Characterization of a Bacillus-Derived Novel Broad-Spectrum Antifungal Lipopeptide Variant against Candida tropicalis and Candida auris and Unravelling Its Mode of Action

Limited treatment options, recalcitrance, and resistance to existing therapeutics encourage the discovery of novel antifungal leads for alternative therapeutics. Antifungal lipopeptides have emerged as potential candidates for developing new and alternative antifungal therapies. In our previous studies, we isolated and identified the lipopeptide variant AF(4) and purified it to homogeneity via chromatography from the cell-free supernatant of Bacillus subtilis. AF(4) was found to have broad-spectrum antifungal activity against more than 110 fungal isolates. In this study, we found that clinical isolates of Candida tropicalis and Candida auris exposed to AF(4) exhibited low MICs of 4 to 8 mg/L. Time-kill assays indicated the in vitro pharmacodynamic potential of AF(4). Biocompatibility assays demonstrated ~75% cell viability at 8 mg/L of AF(4), indicating the lipopeptide’s minimally cytotoxic nature. In lipopeptide-treated C. tropicalis and C. auris cells, scanning electron microscopy revealed damage to the cell surface, while confocal microscopy with acridine orange(AO)/propidium iodide (PI) and FUN-1 indicated permeabilization of the cell membrane, and DNA damage upon DAPI (4′,6-diamidino-2-phenylindole) staining. These observations were corroborated using flow cytometry (FC) in which propidium iodide, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and rhodamine 123 (Rh123) staining of cells treated with AF(4) revealed loss of membrane integrity, increased reactive oxygen species (ROS) production, and mitochondrial membrane dysfunction, respectively. Membrane perturbation was also observed in the 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence study and the interaction with ergosterol was observed by an ergosterol binding assay. Decreased membrane dipole potential also indicated the probable binding of lipopeptide to the cell membrane. Collectively, these findings describe the mode of action of AF(4) against fungal isolates by membrane disruption and ROS generation, demonstrating its antifungal potency. IMPORTANCE C. tropicalis is a major concern for candidiasis in India and C. auris has emerged as a resistant yeast causing difficult-to-treat infections. Currently, amphotericin B (AMB) and 5-flucytosine (5-FC) are the main therapeutics for systemic fungal infections; however, the nephrotoxicity of AMB and resistance to 5-FC is a serious concern. Antifungal lead molecules with low adverse effects are the need of the hour. In this study, we briefly describe the antifungal potential of the AF(4) lipopeptide and its mode of action using microscopy, flow cytometry, and fluorescence-based assays. Our investigation reveals the basic mode of action of the investigated lipopeptide. This lipopeptide with broad-spectrum antifungal potency is apparently membrane-active, and there is a smaller chance that organisms exposed to such a compound will develop drug resistance. It could potentially act as a lead molecule for the development of an alternative antifungal agent to combat candidiasis.