RESUMO
Malassezia spp. are lipid-dependent yeasts that have been related to skin mycobiota and dermatological and systemic diseases. Study of lipid droplets (LDs) is relevant to elucidate the unknown role of these organelles in Malassezia and to gain a broader overview of lipid metabolism in Malassezia. Here, we standardized two protocols for the analysis of LDs in M. pachydermatis and M. globosa. The first describes co-staining for confocal laser-scanning fluorescence microscopy, and the second details extraction and purification of LDs. The double stain is achieved with three different neutral lipid fluorophores, namely Nile Red, BODIPY™ 493/503, and HCS LipidTOX™ Deep Red Neutral, in combination with Calcofluor White. For LD extraction, cell wall rupture is conducted using Trichoderma harzianum enzymes and cycles of vortexing with zirconium beads. LD purification is performed in a three-step ultracentrifugation process. These standardizations will contribute to the study of the dynamics, morphology, and composition of LDs in Malassezia. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Lipid droplet fluorescence staining Basic Protocol 2: Lipid droplet extraction and purification Support Protocol: Malassezia spp. culture conditions.
Assuntos
Malassezia , Hypocreales , Gotículas LipídicasRESUMO
Candida albicans has emerged as a major public health problem in recent decades. The most important contributing factor is the rapid increase in resistance to conventional drugs worldwide. Synthetic antimicrobial peptides (SAMPs) have attracted substantial attention as alternatives and/or adjuvants in therapeutic treatments due to their strong activity at low concentrations without apparent toxicity. Here, two SAMPs, named Mo-CBP3 -PepI (CPAIQRCC) and Mo-CBP3 -PepII (NIQPPCRCC), are described, bioinspired by Mo-CBP3 , which is an antifungal chitin-binding protein from Moringa oleifera seeds. Furthermore, the mechanism of anticandidal activity was evaluated as well as their synergistic effects with nystatin. Both peptides induced the production of reactive oxygen species (ROS), cell wall degradation, and large pores in the C. albicans cell membrane. In addition, the peptides exhibited high potential as adjuvants because of their synergistic effects, by increasing almost 50-fold the anticandidal activity of the conventional antifungal drug nystatin. These peptides have excellent potential as new drugs and/or adjuvants to conventional drugs for treatment of clinical infections caused by C. albicans.