Media matters! Alterations in the loading and release of Histoplasma capsulatum extracellular vesicles in response to different nutritional milieus

Abstract

Histoplasma capsulatum is a dimorphic fungus that most frequently causes pneumonia, but can also disseminate and proliferate in diverse tissues. Histoplasma capsulatum has a complex secretion system that mediates the release of macromolecule-degrading enzymes and virulence factors. The formation and release of extracellular vesicles (EVs) are an important mechanism for non-conventional secretion in both ascomycetes and basidiomycetes. Histoplasma capsulatum EVs contain diverse proteins associated with virulence and are immunologically active. Despite the growing knowledge of EVs from H. capsulatum and other pathogenic fungi, the extent that changes in the environment impact the sorting of organic molecules in EVs has not been investigated. In this study, we cultivated H. capsulatum with distinct culture media to investigate the potential plasticity in EV loading in response to differences in nutrition. Our findings reveal that nutrition plays an important role in EV loading and formation, which may translate into differences in biological activities of these fungi in various fluids and tissues.

Keywords: BHI; Ham's F12; Histoplasma capsulatum; RPMI; USA; culture media; dimorphic fungus; extracellular vesicles; histoplasmosis; lipidomics; metabolomics; proteomics.

Figure 1.. Protein and ergosterol content of EVs from H. capsulatum grown in distinct media.

The graph shows the protein (A) and ergosterol (B) quantifications of EV isolated from each culture supernatant. Graphs represent average and standard deviations for at least 3 independent EV isolations. * = p < 0.05, ** = p < 0.01, by Tukey multiple comparison test.

Figure 2.. Size of EVs cultivated in distinct media by DLS.

After their isolation from medium supernatant, EVs were analyzed by Dynamic Light Scattering. The graph shows average and standard deviation for samples from 3 independent experiments. * = p < 0.05 by ANOVA followed by Tukey’s multiple comparison test.

Figure 3.. EV proteins in different media conditions.

The heatmap shows differential abundance of proteins in EVs produced by H. capsulatum yeast cells cultivated in distinct media. Proteins were grouped based on function. Group 1: Amino acid transport, TCA cycle, intracellular protein transport, phospholipid translocation, and translation. Group 2: DNA repair, lipid metabolism, endocytosis, and mRNA splicing. Group 3: Transcription regulators and protein folding. Group 4: Carbohydrate and polysaccharide metabolism

Figure 4.

Heatmap with lipids significantly altered in EVs released by H. capsulatum cultured under different media.

Figure 5.. Lysophospholipids in EVs from H. capsulatum grown in different media.

This figure shows the abundance of distinct lysophospholipids found in each condition. Significant differences were found between EV-RPMI and EV-BHI. * = p < 0.05 by one-way ANOVA followed by Dunn’s multiple comparisons test.

Figure 6.. Metabolic pathways affected by different media.

The PCA plot (A) illustrates the distinction between the metabolic profiles of EVs from different media. In the heatmap (B) the metabolites detected in EVs from H. capsulatum cultivated in different media. Some of the detected metabolites were depicted (C) to show their variation among EVs from different growth conditions.