PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans
Guowu Hu, … , Yoshinori Ohsumi, Peter R. Williamson
Guowu Hu, … , Yoshinori Ohsumi, Peter R. Williamson
Published February 7, 2008
Citation Information: J Clin Invest. 2008;118(3):1186-1197. https://doi.org/10.1172/JCI32053.
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Research Article AIDS/HIV

PI3K signaling of autophagy is required for starvation tolerance and virulenceof Cryptococcus neoformans

Abstract

Autophagy is a process by which cells recycle cytoplasm and defective organelles during stress situations such as nutrient starvation. It can also be used by host cells as an immune defense mechanism to eliminate infectious pathogens. Here we describe the use of autophagy as a survival mechanism and virulence-associated trait by the human fungal pathogen Cryptococcus neoformans. We report that a mutant form of C. neoformans lacking the Vps34 PI3K (vps34Δ), which is known to be involved in autophagy in ascomycete yeast, was defective in the formation of autophagy-related 8–labeled (Atg8-labeled) vesicles and showed a dramatic attenuation in virulence in mouse models of infection. In addition, autophagic vesicles were observed in WT but not vps34Δ cells after phagocytosis by a murine macrophage cell line, and Atg8 expression was exhibited in WT C. neoformans during human infection of brain. To dissect the contribution of defective autophagy in vps34Δ C. neoformans during pathogenesis, a strain of C. neoformans in which Atg8 expression was knocked down by RNA interference was constructed and these fungi also demonstrated markedly attenuated virulence in a mouse model of infection. These results demonstrated PI3K signaling and autophagy as a virulence-associated trait and survival mechanism during infection with a fungal pathogen. Moreover, the data show that molecular dissection of such pathogen stress-response pathways may identify new approaches for chemotherapeutic interventions.

Authors

Guowu Hu, Moshe Hacham, Scott R. Waterman, John Panepinto, Soowan Shin, Xiaoguang Liu, Jack Gibbons, Tibor Valyi-Nagy, Keisuke Obara, H. Ari Jaffe, Yoshinori Ohsumi, Peter R. Williamson

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Figure 3

Survival of VPS34 strains in a J774.

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Survival of VPS34 strains in a J774. 16 macrophage cell line. (A) Fungal...
16 macrophage cell line. (A) Fungal cells were opsonized with anti-capsular antibody, and 105 cells were incubated with J774.16 macrophages for 30 min, washed extensively to remove non-phagocytosed cells, and at the indicated times, wells were washed with water containing 0.01% SDS and cultured for growth of fungal cells on YPD (n = 3 for each strain at each time point.) (B) Same as in A, but macrophages were incubated with 105 of a 50:50 mixture of WT and vps34Δ mutant cells, and strains were identified from recovered mixtures as described in Methods. (C) Same as in B, but vps34Δ mutant strains were prestained with calcofluor (blue) prior to macrophage inoculation and fungal viability determined by FUN-1 epifluorescence (red) at the indicated times. Blue arrows indicated vps34Δ cells, yellow arrows indicate WT cells. Scale bars: 5 microns. (D) Analysis of viable fungal cells in macrophage by FUN-1 staining. Macrophages containing mixtures of WT (unlabeled, blue bars) and vps34Δ mutant (calcofluor-labeled, yellow bars) cells were identified and fungal cells scored by the presence of FUN-1 staining at the indicated times (*P < 0.001). (E) Macrophages were infected with either WT or vps34Δ mutant fungal cells as in A, and at 3 h, macrophage protein was harvested and subjected to 15% SDS-PAGE, followed by western blot using 1:1000 anti-LC3 antibody and developed using a horseradish peroxidase anti-rabbit antibody as described in Methods. Lane 1 corresponds to macrophages infected with WT cells and lane 2 to macrophages infected with vps34Δ mutant fungal cells.