Abstract
Cryptococcus neoformans (Cn), the major causative agent of human fungal meningoencephalitis, replicates within phagolysosomes of infected host cells. Despite more than a half-century of investigation into host-Cn interactions, host factors that mediate infection by this fungal pathogen remain obscure. Here, we describe the development of a system that employs Drosophila S2 cells and RNA interference (RNAi) to define and characterize Cn host factors. The system recapitulated salient aspects of fungal interactions with mammalian cells, including phagocytosis, intracellular trafficking, replication, cell-to-cell spread and escape of the pathogen from host cells. Fifty-seven evolutionarily conserved host factors were identified using this system, including 29 factors that had not been previously implicated in mediating fungal pathogenesis. Subsequent analysis indicated that Cn exploits host actin cytoskeletal elements, cell surface signaling molecules, and vesicle-mediated transport proteins to establish a replicative niche. Several host molecules known to be associated with autophagy (Atg), including Atg2, Atg5, Atg9 and Pi3K59F (a class III PI3-kinase) were also uncovered in our screen. Small interfering RNA (siRNA) mediated depletion of these autophagy proteins in murine RAW264.7 macrophages demonstrated their requirement during Cn infection, thereby validating findings obtained using the Drosophila S2 cell system. Immunofluorescence confocal microscopy analyses demonstrated that Atg5, LC3, Atg9a were recruited to the vicinity of Cn containing vacuoles (CnCvs) in the early stages of Cn infection. Pharmacological inhibition of autophagy and/or PI3-kinase activity further demonstrated a requirement for autophagy associated host proteins in supporting infection of mammalian cells by Cn. Finally, systematic trafficking studies indicated that CnCVs associated with Atg proteins, including Atg5, Atg9a and LC3, during trafficking to a terminal intracellular compartment that was decorated with the lysosomal markers LAMP-1 and cathepsin D. Our findings validate the utility of the Drosophila S2 cell system as a functional genomic platform for identifying and characterizing host factors that mediate fungal intracellular replication. Our results also support a model in which host Atg proteins mediate Cn intracellular trafficking and replication.
Highlights
Over the past half-century, Cryptococcus neoformans (Cn), an opportunistic encapsulated yeast, has emerged as the major causative agent of fungal meningoencephalitis in humans and animals worldwide [1,2]
The pathogen shows a predilection to the central nervous system (CNS), which can result in life-threatening cryptococcal meningoencephalitis
Our data demonstrate that the combination of Drosophila S2 cells and RNA interference technology provides a powerful platform for identifying and characterizing host factors that mediate Cn infection
Summary
Over the past half-century, Cryptococcus neoformans (Cn), an opportunistic encapsulated yeast, has emerged as the major causative agent of fungal meningoencephalitis in humans and animals worldwide [1,2]. Initial infection in the lungs results from the inhalation of fungal cells from the environment [1,2]. The most common clinical form of systemic Cryptococcus infection in humans is meningoencephalitis. Several Cn features, such as polysaccharide capsule synthesis [4,5], melanin production [6,7], and growth at host physiological temperatures [7] are associated with virulence. Several signaling pathways, including the cAMP-PKA pathway, three MAP kinase pathways (involving Cpk, Hog and Mpk1), the Ras specific pathway, and the Ca2+-calcineurin pathway modulate Cn morphological differentiation, virulence, and stress responses [8]. Numerous Cn virulence factors have been characterized [9]
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