Abstract
Every year, more than 250,000 invasive candidiasis infections are reported with 50,000 deaths worldwide. The limited number of antifungal agents necessitates the need for alternative antifungals with potential novel targets. The 2-benzylidenebenzofuran-3-(2H)-ones have become an attractive scaffold for antifungal drug design. This study aimed to determine the antifungal activity of a synthetic aurone compound and characterize its mode of action. Using the broth microdilution method, aurone SH1009 exhibited inhibition against C. albicans, including resistant isolates, as well as C. glabrata, and C. tropicalis with IC50 values of 4-29 μM. Cytotoxicity assays using human THP-1, HepG2, and A549 human cell lines showed selective toxicity toward fungal cells. The mode of action for SH1009 was characterized using chemical-genetic interaction via haploinsufficiency (HIP) and homozygous (HOP) profiling of a uniquely barcoded Saccharomyces cerevisiae mutant collection. Approximately 5300 mutants were competitively treated with SH1009 followed by DNA extraction, amplification of unique barcodes, and quantification of each mutant using multiplexed next-generation sequencing. Barcode post-sequencing analysis revealed 238 sensitive and resistant mutants that significantly (FDR P values ≤ 0.05) responded to aurone SH1009. The enrichment analysis of KEGG pathways and gene ontology demonstrated the cell cycle pathway as the most significantly enriched pathway along with DNA replication, cell division, actin cytoskeleton organization, and endocytosis. Phenotypic studies of these significantly enriched responses were validated in C. albicans. Flow cytometric analysis of SH1009-treated C. albicans revealed a significant accumulation of cells in G1 phase, indicating cell cycle arrest. Fluorescence microscopy detected abnormally interrupted actin dynamics, resulting in enlarged, unbudded cells. RT-qPCR confirmed the effects of SH1009 in differentially expressed cell cycle, actin polymerization, and signal transduction genes. These findings indicate the target of SH1009 as a cell cycle-dependent organization of the actin cytoskeleton, suggesting a novel mode of action of the aurone compound as an antifungal inhibitor.
Highlights
Life-threatening fungal infections have been increasing due to the difficulties with diagnosis and treatment that accelerate mortality rates associated with fungal infections, which exceed deaths caused by malaria [1]
Chemical-genetic interaction analysis predicted that SH1009 targets cell cycle-dependent organization of the actin cytoskeleton and endocytosis, suggesting a novel mode of action for this aurone compound
Phenotypic studies for these significantly enriched biological responses were completed in C. albicans demonstrating G1 phase-arrested cells with abnormal, depolarized actin cytoskeleton
Summary
Life-threatening fungal infections have been increasing due to the difficulties with diagnosis and treatment that accelerate mortality rates associated with fungal infections, which exceed deaths caused by malaria [1]. Candida albicans is the most frequently isolated opportunistic fungal pathogen and is implicated in superficial mucosal infections, or candidiasis of the oral cavity and genitalia of humans, in immunocompromised patients [2]. With the introduction of antibacterial antibiotics as medical therapy in the 1940s, a gradual increase in the number of invasive candidiasis cases has been reported due to antibiotic-associated loss of the bacterial biota and subsequent colonization of Candida spp. on epithelial surfaces, a requirement for pathogenesis [3]. Several risk factors contribute to the pathogenesis of invasive candidiasis, including organ transplantation, prolonged hospitalization in an intensive care unit, catheterization, and intensive utilization of antibiotics and immunosuppressive agents. These factors could lead Candida spp. to colonize mucosal surfaces, resulting in superficial infections. Certain virulence factors are attributed to the pathogenicity of Candida spp., including adherence to epithelial surfaces, dimorphic growth, biofilm formation, and production of tissue-damaging enzymes [4, 5]
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