Trichophyton rubrumcauses ringworm worldwide. Citral (CIT), extracted fromPectisplants, is a monoterpene and naturally composed of geometric isomers neral (cis-citral) and geranial (trans-citral). CIT has promising antifungal activities and ergosterol biosynthesis inhibition effects against several pathogenic fungi. However, no study has focused on neral and geranial againstT. rubrum, which hinders the clinical application of CIT. This study aimed to compare antifungal activities of neral and geranial and preliminarily elucidate their ergosterol biosynthesis inhibition mechanism againstT. rubrum. Herein, the disc diffusion assays, cellular leakage measurement, flow cytometry, SEM/TEM observation, sterol quantification, and sterol pattern change analyses were employed. The results showed geranial exhibited larger inhibition zones (p< 0.01 or 0.05), higher cellular leakage rates (p< 0.01), increased conidia with damaged membranes (p< 0.01) within 24 h, more distinct shriveled mycelium in SEM, prominent cellular material leakage, membrane damage, and morphological changes in TEM. Furthermore, geranial possessed more promising ergosterol biosynthesis inhibition effects than neral, and both induced the synthesis of 7-Dehydrodesmosterol and Cholesta-5,7,22,24-tetraen-3β-ol, which represented marker sterols when ERG6 was affected. These results suggest geranial is more potent than neral againstT. rubrum, and both inhibit ergosterol biosynthesis by affecting ERG6.Keywords:neral;geranial;Trichophyton rubrum;ergosterol biosynthesis;ERG6;citral;antifungal activity;dermatophytes
Summary Filamentation is an important virulence factor of the pathogenic fungus Candida albicans . The abolition of Candida albicans hyphal formation by disrupting sterol synthesis is an important concept for the development of antifungal drugs with high safety. Here, we conduct a high-throughput screen using a C . albicans strain expressing green fluorescent protein-labeled Dpp3 to identify anti-hypha agents by interfering with ergosterol synthesis. The antipyrine derivative H55 is characterized to have minimal cytotoxicity and potent inhibition of C . albicans hyphal formation in multiple cultural conditions. H55 monotherapy exhibits therapeutic efficacy in mouse models of azole-resistant candidiasis. H55 treatment increases the accumulation of zymosterol , the substrate of C-24 sterol methyltransferase (Erg6). The results of enzyme assays, photoaffinity labeling , molecular simulation, mutagenesis , and cellular thermal shift assays support H55 as an allosteric inhibitor of Erg6. Collectively, H55, an inhibitor of the fungal-specific enzyme Erg6, holds potential to treat C . albicans infections.
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