Background:

Cryptococcus neoformans is a human pathogenic fungus that infects mainly
immunocompromised patients causing cryptococcosis. Among others, one of its
strategies of virulence is the secretion of extracellular vesicles (EVs) containing
important polysaccharides with immunomodulatory characteristics and biomolecules constituents of the capsule assembly. It's known that different serotype of C. neoformans produce different inflammatory responses when incubated with macrophages and, depending on the growth condition, these fungi release metabolites and virulence factors in distinct concentration. Thus, we show that depending on the culture medium composition, C. neoformans products different virulence factors and different extracellular vesicles.

Methods:

We evaluated the characteristics of the extracellular vesicles produced by a
capsular and acapsular serotypes of C. neoformans (B3501 and Cap67) growing in three different concentrations of the culture medium Sabouraud Dextrose Broth (10%, 50% and 100%) or a poor medium (Minimum Medium). These EVs were obtained by a multistep based protocol based on centrifugation, filtering, concentration and ultracentrifugations, as described elsewhere (Rodrigues et al. 2007). Their morphology was characterized by Dynamic Light Scattering (DLS) and Electronic Microscopy and they were quantified by Amplex Red essay for ergosterol quantification and Micro BCA assay for protein quantification. Aiming to have new insights of the influence of these vesicles on the inflammasome activation and the resultant IL-1β production, they were incubated with bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) from C57BL/6 mice and the cytokine of the supernatant was quantified by ELISA assay. Furthermore, in vivo experiments are being conducted to analyze the inflammasome genes activated during the infection with C. neoformans after treatment with the EVs.

Results:

The results show that depending on the nutrient concentration in the culture
medium, C. neoformans produce vesicles with different hydrodynamics sizes and theymodulate the inflammasome by different ways, promoting the production of  the cytokine IL-1 β at varying levels. EVs obtained from a poor medium (Minimum Medium) showed higher hydrodynamic diameters, increased production of virulence factors like Laccase and Urease and more GXM content than EVs obtained from a rich medium, showing that these molecules are important in stress situations.

Conclusion:

Therefore, we can conclude that the culture medium composition is greatly
important to modulate the characteristic of the extracellular vesicles produced by the yeasts. Furthermore, the fact that they present more conventional virulence factors, more protein and a higher size when obtained from a poor medium corroborate with the hypothesis that these vesicles are virulence factors and are different under nutritional stress situations, being important during infection.

4 / Inflammasome modulation by Cryptococcus neoformans extracelular vesicles produced in different conditions