PNNL

Abstract

Diverse pathogenic fungi secrete extracellular vesicles (EV) that contain macromolecules, including virulence factors that can modulate the host immune response. To further evaluate the impact of opsonization on EV loading and consequently its impact on mammalian cells, we performed a proteomic analysis of EV released by Histoplasma capsulatum treated with two different concentrations of protective (6B7) and non-protective (7B6) monoclonal antibodies (mAbs) that bind a heat shock protein (hsp60) located at the fungal cell surface. We found that the mAbs differentially regulate EV content in concentration-dependent and –independent manners. Enzymatic assays demonstrated that laccase activity in EV was reduced after treating with 6B7, but urease activity was not altered by mAb treatment. The uptake of H. capsulatum by macrophages was inhibited when macrophages were exposed to EV 1 hour prior the in vitro challenge, but this suppression did not occur after pre-incubation for 5 or 24 hours with EV Moreover, fungal intracellular proliferation was inhibited in macrophages incubated for 1 hour with EV from 6B7-treated H. capsulatum. In summary, our findings show that opsonization quantitatively and qualitatively modifies how H. capsulatum loads and secretes its EV and how these modifications impact fungal virulence mechanisms, suggesting that EV sorting and secretion are dynamic mechanisms for a fine-tuned response by fungal cells.