Histoplasma capsulatum is the causative agent of histoplasmosis. Treating this fungal infection conventionally has significant limitations, prompting the search for alternative therapies. In this context, fungal extracellular vesicles (EVs) hold relevant potential as both therapeutic agents and targets for the treatment of fungal infections. To explore this further, we conducted a study using pharmacological inhibitors of chitinase (methylxanthines) to investigate their potential to reduce EV release and its subsequent impact on fungal virulence in an in vivo invertebrate model. Our findings revealed that a subinhibitory concentration of the methylxanthine, caffeine, effectively reduces EV release, leading to a modulation of H. capsulatum virulence. To the best of our knowledge, this is the first reported instance of a pharmacological inhibitor that reduces fungal EV release without any observed fungicidal effects.

This study aimed to investigate the effects of cyclosporine on the morphology, cell wall structure, and secretion characteristics of Cryptococcus neoformans. The minimum inhibitory concentration (MIC) of cyclosporine was found to be 2 µM (2.4 µg/mL) for the H99 strain. Yeast cells treated with cyclosporine at half the MIC showed altered morphology, including irregular shapes and elongated projections, without an effect on cell metabolism. Cyclosporine treatment resulted in an 18-fold increase in chitin and an 8-fold increase in lipid bodies, demonstrating changes in the fungal cell wall structure. Cyclosporine also reduced cell body and polysaccharide capsule diameters, with a significant reduction in urease secretion in C. neoformans cultures. Additionally, the study showed that cyclosporine increased the viscosity of secreted polysaccharides and reduced the electronegativity and conductance of cells. The findings suggest that cyclosporine has significant effects on C. neoformans morphology, cell wall structure, and secretion, which could have implications for the development of new antifungal agents.

Sporotrichosis is a fungal infection caused by Sporothrix species, with Sporothrix brasiliensis as a prevalent pathogen in Latin America. Despite its clinical importance, the virulence factors of S. brasiliensis and their impact on the pathogenesis of sporotrichosis are still poorly understood. This study evaluated the morphostructural plasticity of S. brasiliensis, a fungus that causes sporotrichosis. Three cell surface characteristics, namely cell surface hydrophobicity, Zeta potential, and conductance, were assessed. Biofilm formation was also analyzed, with measurements taken for biomass, extracellular matrix, and metabolic activity. In addition, other potential and poorly studied characteristics correlated with virulence such as lipid bodies, chitin, and cell size were evaluated. The results revealed that the major phenotsypic features associated with fungal virulence in the studied S. brasiliensis strains were chitin, lipid bodies, and conductance. The dendrogram clustered the strains based on their overall similarity in the production of these factors. Correlation analyses showed that hydrophobicity was strongly linked to the production of biomass and extracellular matrix, while there was a weaker association between Zeta potential and size, and lipid bodies and chitin. This study provides valuable insights into the virulence factors of S. brasiliensis and their potential role in the pathogenesis of sporotrichosis.

Candida auris is a globally emerging pathogenic fungus described in Japan in 2009. This fungus has been identified mainly in nosocomial environments, associated with a high frequency of multidrug-resistant strains, and mortality rates reach 60%. C. auris is divided into 6 biogeographic clades, and there is a correlation between the clades and resistance against antifungals. In the current report, 8 strains of C. auris isolated in the Montefiore Medical Center, Bronx were analyzed to assess their clade (via ClaID) and common molecular determinants of antifungal resistance. We assessed antifungal resistance as well as the efficacy of a novel nitric oxide-donating microparticle as an alternative approach against C. auris in vitro through microplate susceptibility tests. Virulence was also determined in a Galleria mellonella model. Our results indicate that 7 out of 8 strains, belonging to clade 1, were resistant to fluconazole, while clade 2 was susceptible. Additionally, the clade 2 strain was more susceptible to treatment with the microparticle, while also being more virulent in an invertebrate model of infection. Our findings were then correlated to visualize parallels between clade identification and resistance/virulence patterns.

Extracellular vesicles (EVs) released by Candida albicans are multi-antigenic compartments considered as promising prototypes for vaccine development. However, their stability, appropriate storage, and handling conditions are largely unexplored, which raises questions related to their biotechnological applicability. Here, we evaluated the physical and functional stability of C. albicans EVs under long-term storage. Furthermore, we conducted a comparative analysis of these properties in C. albicans EVs obtained through three commonly utilized isolation protocols documented in the literature. After identifying the most efficient isolation method for optimal yield, we devised a potential quality control for EV isolation based on protein and sterol ratios. Subsequently, we investigated the impact of drying EVs using vacuum centrifugation at room temperature or −4°C and the effect of freeze-thaw cycles on EV stability. Transmission electron microscopy (TEM) revealed that EVs maintained morphological stability after long-term (up to 4 years) storage at −80°C, as well as storage at room temperature, 4°C, and −20°C for 7 days with or without vacuum centrifugation, with a tendency of higher recovery when a lower temperature is used. Remarkably, all of the C. albicans EV suspensions maintained their biological properties as demonstrated by their ability to protect Galleria mellonella against C. albicans infection. However, the number of freeze-thaw cycles significantly impacted the protective effect of the EVs. Overall, our findings demonstrate that C. albicans EVs maintain notable morphological and biological stability under several conditions, enabling their efficient and reproducible utilization in research and potentially as therapeutic agents. IMPORTANCE: Extracellular vesicles (EVs) released by Candida albicans are promising vaccine prototypes due to their multi-antigenic nature. However, their storage and handling conditions are not well understood, raising concerns about their biotechnological use. This study evaluated the long-term physical and functional stability of C. albicans EVs. We compared three isolation methods to identify the most effective one and suggested a quality control measure based on protein and sterol ratios. We also examined the effects of vacuum drying and freeze-thaw cycles on EV stability. Our findings show that C. albicans EVs maintain their biological function after long-term storage at −80°C and under various conditions. Notably, their protective effect in an insect model was reduced through repeated freeze-thaw cycles. This research provides valuable insights for the efficient use of these vesicles in future studies.