Protocol for separation of fungal extracellular vesicles using ultracentrifugation from solid medium cultures.

Extracellular vesicles (EVs) have been identified in diverse fungi, including human pathogens. In this protocol, we present two techniques for isolating and analyzing fungal EVs. The first is for high-throughput screening, and the second is for yielding concentrated samples suitable for centrifugation-based density gradients. We describe steps for analytical assays such as nano-flow cytometry and nanoparticle tracking analysis to measure EV dimensions and concentration. EV suspensions can serve diverse assays, including electron microscopy, compositional determination, and cell-to-cell communication assays. For complete details on the use and execution of this protocol, please refer to Rizzo et al.,1 Rizzo et al.,2 Reis et al.,3 and Reis et al.4.

Transcriptomic analysis reveals that mTOR pathway can be modulated in macrophage cells by the presence of cryptococcal cells.

Cryptococcus neoformans and Cryptococcus gattii are the etiological agents of cryptococcosis, a high mortality disease. The development of such disease depends on the interaction of fungal cells with macrophages, in which they can reside and replicate. In order to dissect the molecular mechanisms by which cryptococcal cells modulate the activity of macrophages, a genome-scale comparative analysis of transcriptional changes in macrophages exposed to Cryptococcus spp. was conducted. Altered expression of nearly 40 genes was detected in macrophages exposed to cryptococcal cells. The major processes were associated with the mTOR pathway, whose associated genes exhibited decreased expression in macrophages incubated with cryptococcal cells. Phosphorylation of p70S6K and GSK-3ß was also decreased in macrophages incubated with fungal cells. In this way, Cryptococci presence could drive the modulation of mTOR pathway in macrophages possibly to increase the survival of the pathogen.

The paradoxical and still obscure properties of fungal extracellular vesicles.

Early compositional studies of fungal EVs revealed a complex combination of biomolecules, including proteins, lipids, glycans, polysaccharides, nucleic acid and pigments, indicating that these compartments could be involved with multiple functions. Curiously, some of the activities attributed to fungal EVs were already attested experimentally and are implicated with contrasting effects in vitro and in vivo. For instance, the presence of virulence factors is correlated with increased pathogenic potential. Indeed, the administration to hosts of EVs along with some fungal pathogens seems to help the disease development. However, it has been clearly shown that immunization of insects and mice with fungal EVs can protect these animals against a subsequent infection. Fungal EVs not only influence the host response, as concluded from the observation that these compartments also work as messengers between fungal organisms. In this context, despite their size characterization, other physical properties of EVs are poorly known. For instance, their stability and half-life under physiological conditions can be a crucial parameter determining their long-distance effects. In this review, we will discuss the paradoxical and still unexploited functions and properties of fungal EVs that could be determinant for their biological functions.

Fungal Extracellular Vesicles as a Potential Strategy for Vaccine Development.

Extracellular vesicles (EVs) are released by virtually all live cells. In fungal organisms, the EVs traverse the cell wall and reach the extracellular environment, where they can interact with host cells and potentially impact the disease outcome. Compositional analyses have demonstrated that fungal EVs carry lipids, proteins, polysaccharides, glycans, nucleic acids, and a diversity of small metabolites. Among this variety of compounds, several molecules with immunogenic properties were characterized. It corroborates with their ability to stimulate innate immune cells, induce antibody production and protect insects and mice against fungal infections. In this chapter, we discuss the advantages of using fungal EVs as a new platform for the development of antifungal vaccines.

The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells.

The pathogenic yeast Cryptococcus neoformans secretes numerous proteins, such as heat shock proteins, by unconventional mechanisms during its interaction with host cells. Hsp70 is a conserved chaperone that plays important roles in various cellular processes, including the interaction of fungi with host immune cells. Here, we report that sera from individuals with cryptococcosis infection recognize a recombinant C. neoformans Hsp70 (Cn_rHsp70). Moreover, immunofluorescence assays using antibodies against Cn_rHsp70 revealed the localization of this protein at the cell surface mainly in association with the capsular network. We found that the addition of Cn_rHsp70 positively modulated the interaction of C. neoformans with human alveolar epithelial cells and decreased fungal killing by mouse macrophages, without affecting phagocytosis rates. Immunofluorescence analysis showed that there was a competitive association among the receptor, GXM and Cn_rHsp70, indicating that the Hsp70-binding sites in host cells appear to be shared by glucuronoxylomannan (GXM), the major capsular antigen in C. neoformans. Our observations suggest additional mechanisms by which Hsp70 influences the interaction of C. neoformans with host cells.