Training in manipulation and culture of Cryptococcus sp: methods for analyzing cell wall components, purification of extracellular vesicles, and experimental infection in Galleria mellonella

• Grant number: 24/17879-0
• Support Opportunities: Scholarships in Brazil - Program to Stimulate Scientific Vocations
• Start date: February 17, 2025
• End date: April 05, 2025
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Marcio Lourenço Rodrigues
• Grantee: Mariana Eberle dos Santos
• Host Institution: Instituto Carlos Chagas. Fundação Oswaldo Cruz (Fiocruz). Ministério da Saúde (Brasil)

Abstract

The undergrad project of the student Mariana Eberle aims to study primary metabolism, particularly lipid metabolism, in Paracoccidioides brasiliensis. Preliminary results from the originating group, in which proteomics of yeasts were performed after 24 hours of calcineurin inhibition- a calcium- and calmodulin-dependent phosphatase- using cyclosporin A (CSA), suggest that the fungus undergoes vigorous metabolic reprogramming. This hypothesis is based on the fact that there is an alteration in the pattern of lipid droplets in the cells, which decrease in size, suggesting that the yeasts need to utilize this carbon source to adapt to stress. At the same time, enzymes involved in lipid, protein, and carbohydrate metabolism were identified with their relative abundance altered by calcineurin inhibition, apparently compromising glycolysis, gluconeogenesis, the citric acid cycle, the glyoxylate cycle, and beta-oxidation.Additionally, although these are empirical observations made during protein extraction experiments, yeasts treated with CSA need to be centrifuged at higher speeds and for longer periods than untreated yeasts. They also appear to have increased rigidity in their cell walls during mechanical lysis, suggesting alterations in cell wall components. The hypothesis is that the metabolic reprogramming observed with calcineurin inhibition, by affecting primary carbohydrate metabolism, leads to alterations in glycan components of the cell wall. Thus, considering the expertise and history of Prof. Márcio Rodrigues' group in glycobiology, the goal and work plan for this training internship propose learning techniques for analyzing cell wall components in the Cryptococcus model. With this, the student is also expected to receive training in handling and maintaining Cryptococcus cultures, as well as techniques for isolating extracellular vesicles (EV) from this fungus, to later apply in P. brasiliensis.Upon returning to the original laboratory, in addition to analyzing cell wall components in the P. brasiliensis model, the aim is to continue evaluating the glycan content and enzymes involved in remodeling cell wall components in EVs from mycelia and yeasts treated with different concentrations of CSA. Furthermore, the student intends to learn techniques for maintaining Galleria mellonella colonies for future infection with P. brasiliensis as a model to test the action of cell wall components and glycan content of purified EVs from fungi on virulence and infection in this model.This learning experience will contribute to the student's future master's project, which will focus on studying aspects of primary carbon metabolism related to the fungus's adaptation to stress conditions, such as those caused by calcineurin inhibition. Since calcineurin is associated with stress adaptability in different fungi and given the belief that the ability to adapt to the host's internal environment is what ensures the success of infection, demonstrating a possible role of calcineurin in controlling the content and/or release of EVs could reveal important molecules (cell wall remodeling enzymes and glycans) for virulence. (AU)