Vesicular transport in Histoplasma capsulatum: an effective mechanism for trans-cell wall transfer of proteins and lipids in ascomycetes

Abstract

Vesicular secretion of macromolecules has recently been described in the basidiomycete Cryptococcus neoformans, raising the question as to whether ascomycetes similarly utilize vesicles for transport. In the present study, we examine whether the clinically important ascomycete Histoplasma capsulatum produce vesicles and utilized these structures to secrete macromolecules. Transmission electron microscopy (TEM) shows transcellular secretion of vesicles by yeast cells. Proteomic and lipidomic analyses of vesicles isolated from culture supernatants reveal a rich collection of macromolecules involved in diverse processes, including metabolism, cell recycling, signalling and virulence. The results demonstrate that H. capsulatum can utilize a trans-cell wall vesicular transport secretory mechanism to promote virulence. Additionally, TEM of supernatants collected from Candida albicans, Candida parapsilosis, Sporothrix schenckii and Saccharomyces cerevisiae documents that vesicles are similarly produced by additional ascomycetes. The vesicles from H. capsulatum react with immune serum from patients with histoplasmosis, providing an association of the vesicular products with pathogenesis. The findings support the proposal that vesicular secretion is a general mechanism in fungi for the transport of macromolecules related to virulence and that this process could be a target for novel therapeutics.

Fig.1

TEM of extracellular vesicles obtained by ultracentrifugation of culture supernatants from Histoplasma capsulatum showing bilayered membranes and different profiles of electron density. Bars, 100 nm (B, C and E) and 200 nm (A, D and F).

Fig.2

Size analysis of vesicles from H. capsulatum. Five hundred and eight vesicles were analyzed and the size ranged from 10 to 350 nm.

Fig.3

Vesiclular structures were observed in association with the cell wall (A, C and D) and the extracellular environment (B).

Fig.4

Lipid analysis by mass spectrometry of H. capsulatum vesicular components. Total phospholipids were fractionated by silica gel 60 chromatography and analyzed by ESI-MS, in negative- (A) or positive-ion (B) mode. The ion species corresponding to major phospholipids are indicated. These ions were subjected to MS-MS analysis, allowing the identification of 18 phospholipids (Table 1; Supplemental Figure 1). m/z, mass to charge ratio.

Fig. 5

TEM of extracellular vesicles from S. cerevisiae (A, B), C. parapsilosis (C, D), S. schenckii (E, F) and C. albicans (G, H). The structures identified were similar to vesicles produced by C. neoformans and H. capsulatum. Bars 100 nm.

Fig. 6

H. capsulatum vesicles contain immunoreactive proteins. Pooled serum from patients with histoplasmosis reacts with proteins from extracellular H. capsulatum vesicles. Lane A shows the molecular weight marker. Lane B shows H. capsulatum pooled hyperimmune sera reacting with extracts from H. capsulatum vesicles, whereas the non-immune serum in lane C does not interact with the extracted proteins. Lanes D and E demonstrate the binding of monoclonal antibodies to histone 2B (17 kDa; corresponding to *, lane B) and heat shock protein 60 (62 kDa; corresponding to ♦, lane B), respectively, in the vesicular protein preparations.