Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence

Abstract

Cryptococcus neoformans produces vesicles containing its major virulence factor, the capsular polysaccharide glucuronoxylomannan (GXM). These vesicles cross the cell wall to reach the extracellular space, where the polysaccharide is supposedly used for capsule growth or delivered into host tissues. In the present study, we characterized vesicle morphology and protein composition by a combination of techniques including electron microscopy, proteomics, enzymatic activity, and serological reactivity. Secretory vesicles in C. neoformans appear to be correlated with exosome-like compartments derived from multivesicular bodies. Extracellular vesicles manifested various sizes and morphologies, including electron-lucid membrane bodies and electron-dense vesicles. Seventy-six proteins were identified by proteomic analysis, including several related to virulence and protection against oxidative stress. Biochemical tests indicated laccase and urease activities in vesicles. In addition, different vesicle proteins were recognized by sera from patients with cryptococcosis. These results reveal an efficient and general mechanism of secretion of pathogenesis-related molecules in C. neoformans, suggesting that extracellular vesicles function as "virulence bags" that deliver a concentrated payload of fungal products to host effector cells and tissues.

FIG. 1.

Electron microscopic appearance (left panels) and prevalence (right panels) of the four major vesicle morphological groups observed in preparations of extracellular vesicles from C. neoformans. The total population analyzed consisted of 419 different vesicles. Scale bars, 200 nm.

FIG. 2.

Laccase (A), urease (B), and phosphatase (C) activities are associated with extracellular vesicles in C. neoformans. (A). Vesicles purified from culture supernatants of strains H99 and the serotype D laccase mutants 2E-TUC (complemented strain) and 2E-TU (LAC1 deletion strain) were incubated in the presence of l-DOPA and analyzed spectrophotometrically. Vesicles purified from the supernatants of strain H99 were also incubated in urease (B) and phosphatase (C) reaction media, followed by spectrophotometric determination of enzyme activity.

FIG. 3.

Vesicle-associated proteins are recognized by sera from cryptococcosis patients. Vesicle-associated proteins (a and b) were separated by SDS-PAGE and incubated with pooled sera from healthy individuals (a) or cryptococcosis patients (b). Molecular masses for standard (left values) or vesicle (right values) proteins are indicated.

FIG. 4.

Functional classification of the C. neoformans vesicle proteins. The number of proteins found for each class is shown. Unidentified proteins are not shown. For details, see Table S1 in the supplemental material.

FIG. 5.

TEM of C. neoformans suggesting the presence of cytoplasmic vacuole-containing vesicles reminiscent of exosome-like structures. (A) Overview of a C. neoformans cell with different cytoplasmic vacuoles containing vesicles (black asterisks). The white asterisk indicates the cell wall. Scale bar, 500 nm. A magnified view of the vesicle-containing vacuoles is shown. Panel B demonstrates that these structures are surrounded by a bilayered membrane, which sometimes invaginates (arrow). A close association with the cell wall (white asterisk) was observed, suggesting fusion with the plasma membrane. Scale bar, 200 nm. (C) Intracellular and extracellular vesicles (black arrows) have similar dimensions. Scale bar, 200 nm.