A novel protocol for the isolation of fungal extracellular vesicles reveals the participation of a putative scramblase in polysaccharide export and capsule construction in Cryptococcus gattii.

Abstract

22Regular protocols for the isolation of fungal extracellular vesicles (EVs) are time-consuming, 23 hard to reproduce, and produce low yields. In an attempt to improve the protocols used for 24 EV isolation, we explored a model of vesicle production after growth of Cryptococcus gattii 25 and C. neoformans on solid media. Nanoparticle tracking analysis in combination with 26 transmission electron microscopy revealed that C. gattii and C. neoformans produced EVs in 27 solid media. These results were reproduced with an acapsular mutant of C. neoformans, as 28 well as with isolates of Candida albicans, Histoplasma capsulatum, and Saccharomyces 29 cerevisiae. Cryptococcal EVs produced in solid media were biologically active and contained 30 regular vesicular components, including the major polysaccharide glucuronoxylomannan 31 (GXM) and RNA. Since the protocol had higher yields and was much faster than the regular 32 methods used for the isolation of fungal EVs, we asked if it would be applicable to address 33 fundamental questions related to cryptococcal secretion. On the basis that polysaccharide 34 export in Cryptococcus requires highly organized membrane traffic culminating with EV 35 release, we analyzed the participation of a putative scramblase (Aim25, CNBG_3981) in EV-36 mediated GXM export and capsule formation in C. gattii. EVs from a C. gattii aim25 strain 37 differed from those obtained from wild-type (WT) cells in physical-chemical properties and 38 cargo. In a model of surface coating of an acapsular cryptococcal strain with vesicular GXM, 39EVs obtained from the aim25 mutant were more efficiently used as a source of capsular 40 polysaccharides. Lack of the Aim25 scramblase resulted in disorganized membranes and 41 increased capsular dimensions. These results associate the description of a novel protocol 42 for the isolation of fungal EVs with the identification of a previously unknown regulator of 43 polysaccharide release. 44 45 IMPORTANCE. Extracellular vesicles (EVs) are fundamental components of the physiology of 46 cells from all kingdoms. In pathogenic fungi, they participate in important mechanisms of 47 results describe a fast and reliable method for the study of fungal EVs and reveal the 53 participation of scramblase, a phospholipid translocating enzyme, in secretory processes of 54 C. gattii. 55 56 57

Extracellular Vesicle Formation in Cryptococcus deuterogattii Impacts Fungal Virulence and Requires the NOP16 Gene

“…TEM revealed that WT and mutant cells produced EVs with their typical morphological aspects, including cup-shaped structures and bilayered membranes. NTA demonstrated that in the 3 strains, the EV population was mainly distributed in the 100-300 nm range, as extensively observed for the Cryptococcus genus (5,32,33).…”

Extracellular vesicle formation inCryptococcus deuterogattiiimpacts fungal virulence and requires theNOP16gene

“…TEM revealed that WT and mutant cells produced EVs with their typical morphological aspects, including cup-shaped structures and bilayered membranes. NTA demonstrated that in the 3 strains, the EV population was mainly distributed in the 100-300 nm range, as extensively observed for the Cryptococcus genus (5, 32, 33). Minor populations in the 300-600 nm range were also observed in the three strains.…”

The Benefits of Exporting: Engineered Extracellular Vesicles as Promising Vaccine Candidates against Enteric Fever

“…Similar immunological properties were demonstrated for Gram-positive bacteria (17), fungi (18)(19)(20), helminths (21), protozoa (22), and even cancer cells (23). The use of EVs as vaccine prototypes is, therefore, of great interest to the field of vaccinology, and this has motivated the search for improved protocols for the efficient isolation of EVs (24,25).…”