Phagosomal f-actin retention by cryptococcus gattii induces dendritic cell immunoparalysis

Abstract

Cryptococcus gattii is a major cause of life-threatening mycosis in immuno-competent individuals and responsible for the ongoing epidemic outbreak of cryptococ-cosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concom-itant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolu-tion structured illumination microscopy (SR-SIM) revealed that the persistent phago-somal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit pha-gosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also pro-moted DC costimulatory maturation and robust T cell activation and proliferation. Collec-tively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity. IMPORTANCE Cryptococcus yeast species typically display characteristics of opportu-nistic pathogens, with the exception of C. gattii, which can cause life-threatening re-spiratory and disseminated brain infections in otherwise healthy people. The patho-genesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.