Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

Abstract

bCryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to "trap" vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.

Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology, Cargo, and Immunostimulatory Properties

“…We identified between 179 and 278 unique proteins per strain. As with previous extracellular vesicle proteomics analyses, the proteins identified were associated with a wide variety of cellular functions and localizations (10,13,15,21,22). The 179 proteins identified in the wild-type EVs were in line with previously described C. albicans proteins (11).…”

“…Again, the wild-type sample contained a variety of vesicle sizes and densities, as previously reported (10,15,20) (Fig. 3c).…”

“…We first imaged the surface of EVs using scanning electron microscopy (SEM), which revealed a variety of vesicle sizes, consistent with EV produced in other fungal species (10,15) (Fig. 3a).…”

“…EVs were prepared from 3-day-old 100-ml YPD cultures as previously described (11,15). Briefly, cells were removed by centrifugation followed by filtration with a 0.45-m filter.…”

Challenges posed by extracellular vesicles from eukaryotic microbes

“…Interestingly, the absence of ESCRT machinery does not drastically effect the remaining S. cerevisiae EV protein composition [23]. Visual confirmation of this process for C. neoformans and T. cruzi [19,27] was later made via electron microscopy. Ubiquitin machinery, known to stimulate formation of ILV within an MVB, has also been found associated with EV [11,16,27], and therefore may play a broader biological role than solely lysosomal targeted vesicle cargo selection.…”