An Antifungal Mechanism of Protolichesterinic Acid from the Lichen Usnea albopunctata Lies in the Accumulation of Intracellular ROS and Mitochondria-Mediated Cell Death Due to Apoptosis in Candida tropicalis

Abstract

Candida species causes superficial and life-threatening systemic infections and are difficult to treat due to the resistance of these organism to various clinically used drugs. Protolichesterinic acid is a well-known lichen compound. Although the antibacterial activity of protolichesterinic acid has been reported earlier, the antifungal property and its mechanism of action are still largely unidentified. The goal of the present investigation is to explore the anticandidal activity and mechanism of action of protolichesterinic acid, especially against Candida tropicalis. The Minimum Inhibitory Concentration (MIC) value was established through microdilution techniques against four Candida species and out of four species tested, C. tropicalis showed a significant effect (MIC: 2 μg/ml). In the morphological interference assay, we observed the enhanced inhibition of hyphae when the cells were treated with protolichesterinic acid. Time-kill assay demonstrated that the maximum rate of killing was recorded between 2 and 6 h. C. tropicalis exposed to protolichesterinic acid exhibited an increased ROS production, which is one of the key factors of fungal death. The rise in ROS was due to the dysfunction of mitochondria caused by protolichesterinic acid. We confirmed that protolichesterinic acid-induced dysfunction of mitochondria in C. tropicalis. The damage of cell membrane due to protolichesterinic acid treatment was confirmed by the influx of propidium iodide and was further confirmed by the release of potassium ions. The treatment of protolichesterinic acid also triggered calcium ion signaling. Moreover, it commenced apoptosis which is clearly evidenced by Annexin V and propidium iodide staining. Interestingly protolichesterinic acid recorded excellent immunomodulatory property when tested against lymphocytes. Finally protolichesterinic acid showed low toxicity toward a normal human cell line Foreskin (FS) normal fibroblast. In in vivo test, protolichesterinic acid significantly enhanced the survival of C. tropicalis infected Caenorhabditis elegans. This investigation proposes that the protolichesterinic acid induces apoptosis in C. tropicalis via the enhanced accumulation of intracellular ROS and mitochondrial damage, which leads fungal cell death via apoptosis. Our work revealed a new key aspect of mechanisms of action of protolichesterinic acid in Candida species. This article is the first study on the antifungal and mechanism of action of protolichesterinic acid in Candida species.

(E)-2-Hexenal, as a Potential Natural Antifungal Compound, Inhibits Aspergillus flavus Spore Germination by Disrupting Mitochondrial Energy Metabolism

“…Mitochondria play an essential role in the life cycle of cells, including ATP synthesis, calcium mobilization, and apoptosis-programmed cell death . Thus, similar to the plasma membrane, the mitochondria are another potential antifungal target. − Furthermore, several previous studies have demonstrated that mitochondria-mediated apoptosis occurs not only in multicellular organisms but also in single-cell organisms such as fungi, , and the antifungal mechanisms associated with the form of apoptosis triggered by mitochondrial dysfunction and disruption have been proposed. − In this study, it was found that PS externalization as an early marker of apoptosis occurred in the A. flavus spores inhibited by ( E )-2-hexenal. However, systematic studies on the apoptosis of A. flavus spores induced by ( E )-2-hexenal need to be further investigated, including mitochondrial membrane potential, cytochrome c release, intracellular Ca 2+ levels, activation of metacaspases, and DNA damage.…”

Elemental Analysis and In Vitro Evaluation of Antibacterial and Antifungal Activities of Usnea barbata (L.) Weber ex F.H. Wigg from Călimani Mountains, Romania

“…Kumar et al (2017) extracted protolichesterinic acid from Usnea albopunctata and studied its antifungal effect against four Candida species, including C. albicans and C. parapsilosis; the diameters of inhibition zones were 21 mm and 22 mm, respectively [ 78 ]. Our results also showed that the antifungal activity of usnic acid was higher against C. parapsilosis (20 mm) than against C. albicans (10 mm).…”