Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall (original) (raw)
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A role for vesicular transport of macromolecules across cell walls in fungal pathogenesis
Communicative & integrative biology, 2008
In our recent work, we have shown that fungal species from different phyla produce extracellular vesicles. The vesicles are heterogeneous and morphologically similar to mammalian exosomes, with intact bilayered membranes. Proteomic analyses reveal that the vesicles contain a broad array of macromolecules, many of which are associated with fungal virulence. Further, the biological import of the extracellular fungal vesicles is supported by their presence during murine cryptococcosis and the immunoreactivity of convalescent serum from patients with Cryptococcus neoformans or Histoplasma capsulatum vesicle protein extracts.In contrast to most eukaryotic cells, fungi have complex cell walls, that could in theory provide a significant barrier to the secretion of large molecules. The discovery of trans-cell wall vesicular transport in fungi provides a solution to the problem of extracellular transport of macromolecules. Identifying similar vesicles in ascomycetes and basidiomycetes sugges...
Eukaryotic Cell, 2007
The mechanisms by which macromolecules are transported through the cell wall of fungi are not known. A central question in the biology of Cryptococcus neoformans, the causative agent of cryptococcosis, is the mechanism by which capsular polysaccharide synthesized inside the cell is exported to the extracellular environment for capsule assembly and release. We demonstrate that C. neoformans produces extracellular vesicles during in vitro growth and animal infection. Vesicular compartments, which are transferred to the extracellular space by cell wall passage, contain glucuronoxylomannan (GXM), a component of the cryptococcal capsule,
Extracellular Vesicles in the Fungi Kingdom
International Journal of Molecular Sciences, 2021
Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and ...
bioRxiv (Cold Spring Harbor Laboratory), 2023
Cryptosporidium parvum is a common cause of a zoonotic disease and a main cause of diarrhea in newborns around the world. Effective drugs or vaccines are still lacking. Oocyst is the infective form of the parasite; after the ingestion the oocyst excysts and releases four sporozoites into the intestine that rapidly attack the enterocytes. The membrane protein CpRom1 is a large rhomboid protease that is expressed by sporozoites and recognized as antigen by the host immune system. In this study, we observed the release of CpRom1 with extracellular vesicles (EVs) not previously described. To investigate this phenomenon, we isolated and resolved EVs from the excystation medium by differential ultracentrifugation. By fluorescence flow cytometry and transmission electron microscopy (TEM), we identified two types of sporozoite-derived vesicles: large extracellular vesicles (LEVs) and small extracellular vesicles (SEVs), having different a mean size of 150 nm and 60 nm, respectively. Immunodetection experiments proved the occurrence of CpRom1 and the Golgi protein CpGRASP in LEVs, while immune-electron microscopy experiments demonstrated localization of CpRom1 on LEVs surface. TEM and scanning electron microscopy (SEM) showed the generation of LEVs by budding of the outer membrane of sporozoites; conversely, the origin of SEVs remained uncertain. Differences between LEVs and SEVs were observed for protein composition as proved by the corresponding electrophoretic profiles. Indeed, a dedicated proteomic analysis identified 5 proteins unique to LEVs composition and 16 proteins unique to SEVs. Overall, 60 proteins were identified in the proteome of both types of vesicles and most of these proteins (48 in number) were already identified in the molecular cargo of extracellular vesicles from other organisms. Noteworthy, we identified 12 proteins unique to Cryptosporidium spp. that had never been associated with EVs. This last group included the immunodominant parasite antigen glycoprotein GP60, which is one of the most abundant proteins in both LEVs and SEVs.
Lipophilic Dye Staining of Cryptococcus neoformans Extracellular Vesicles and Capsule
Eukaryotic Cell, 2009
Cryptococcus neoformans is an encapsulated yeast that causes systemic mycosis in immunosuppressed individuals. Recent studies have determined that this fungus produces vesicles that are released to the extracellular environment both in vivo and in vitro. These vesicles contain assorted cargo that includes several molecules associated with virulence and implicated in host-pathogen interactions, such as capsular polysaccharides, laccase, urease, and other proteins. To date, visualization of extracellular vesicles has relied on transmission electron microscopy, a time-consuming technique. In this work we report the use of fluorescent membrane tracers to stain lipophilic structures in cryptococcal culture supernatants and capsules. Two dialkylcarbocyanine probes with different spectral characteristics were used to visualize purified vesicles by fluorescence microscopy and flow cytometry. Dual staining of vesicles with dialkylcarbocyanine and RNAselective nucleic acid dyes suggested that a fraction of the vesicle population carried RNA. Use of these dyes to stain whole cells, however, was hampered by their possible direct binding to capsular polysaccharide. A fluorescent phospholipid was used as additional membrane tracer to stain whole cells, revealing punctate structures on the edge of the capsule which are consistent with vesicular trafficking. Lipophilic dyes provide new tools for the study of fungal extracellular vesicles and their content. The finding of hydrophobic regions in the capsule of C. neoformans adds to the growing evidence for a structurally complex structure composed of polysaccharide and nonpolysaccharide components.
Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, hard to reproduce, and produce low yields. In an attempt to improve the protocols used for EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattiiandC. neoformanson solid media. Nanoparticle tracking analysis in combination with transmission electron microscopy revealed that C. gattiiandC. neoformansproduced EVs in solid media. These results were reproduced with an acapsular mutant of C. neoformans, as well as with isolates of Candida albicans, Histoplasma capsulatum, andSaccharomyces cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained regular vesicular components, including the major polysaccharide glucuronoxylomannan (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular methods used for the isolation of fungal EVs, we asked if it would be applicable to address fundamental questi...