Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans

Abstract

Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.

Fig 1. Protein-protein interaction using the two-hybrid assay.

Atg8 protein of C. neoformans interacts with Atg3, Atg4 and Atg7. Growth of Y2HGold yeast strain co-transformed with the indicated bait (pGBKT7) and prey (pGADT7). Two independent clones were tested in a total of n = 2 independent experiments. Plates incubated for 5 days at 30°C. Assay performed with the Matchmaker^™ Gold Yeast Two-Hybrid System (Clontech). DDO: SD/-Leu/-Trp; QDO: SD/-Ade/-His/-Leu/-Trp; X: 40 μg/mL X-α-Gal; A: 200 ng/mL Aureobasidin A. Pair of plasmids pGBKT7-53 and pGADT7-T were used as positive control; and pGBKT7-Lam and pGADT7-T were used as negative controls.

Fig 2. Complementation of the S. cerevisiae atg4Δ and atg8Δ mutants by the respective C. neoformans genes.

Growth analysis after 20 days of incubation in SD/-N/-AA broth at 30°C and 150 rpm. Following nutritional deprivation, serial dilutions of the cell suspension were spotted on YPD agar. Cell viability was checked after 48 hours of incubation at 30°C; n = 3 independent experiments. BY4741 strain was used as a growth control. The cDNA sequences of the ATG4 and ATG8 genes of C. neoformans were cloned into the vector pYES2 (Invitrogen), CnATG4 and CnATG8 respectively.

Fig 3. Starvation challenge for C. neoformans atg4 and atg8 mutants.

Growth sensitivity analysis after 20 days of incubation in SD/-N/-AA broth at 30°C and 150 rpm. Following nutritional deprivation, serial dilutions of the cell suspension were spotted on YPD agar. Cell viability was checked after 48 hours of incubation at 30°C; n = 3 independent experiments. KN99α wild type was used as a growth control.

Fig 4. Atg8 vacuolar degradation during autophagy in C. neoformans.

(A) Wild type (KN99α) and (B) atg4 expressing GFP-Atg8 were grown in a YPD and SD/-N/-AA (nitrogen starvation) broth from 0–4 h. Western blot analysis incubated with primary antibody anti-GFP (1:2,000); n = 3 independent experiments. 41 kDa: full-length Atg8 fused with GFP. 27 kDa: free GFP generated by Atg8 vacuolar proteolysis. Histone was used as an internal reference (anti-histone, 1:2,000).

Fig 5. GFP-Atg8 localization by fluorescence microscopy.

KN99α+GFP-Atg8 and atg4+GFP-Atg8 strains were cultured in YPD and SD/-N/-AA at 30°C and 37°C for 4 h. Vacuole labeled with FM4-64 (Invitrogen) dye. (A) GFP-Atg8 localization during autophagy pathway. White arrows indicate the vacuole membrane. Scale bars represent 5 μm. (B) Percentage of GFP-Atg8 localization at 30°C. (C) Percentage of GFP-Atg8 localization at 37°C. Images were captured with 100× objective lens. n = 3 independent experiments. Error bars represent the standard deviation. Statistical analysis performed using two-way ANOVA: *** (P < 0.001); ns (not significant).

Fig 6. Biological impact of ATG4 (CNAG_02662) and ATG8 (CNAG_00816) gene deletion on C. neoformans.

Analysis of non-autophagic functions for KN99α (wild type); atg4 (two independent mutants); atg8; atg8+ATG8. (A) Oxidative stress phenotype. Growth analysis on YPD supplemented with 2.5–4 mM H₂O₂. Yeast cells were spotted by 10-fold serial dilutions. Plates were incubated at 30°C and 37°C for 48 h. (B) Cell wall integrity phenotype. Growth analysis on YPD supplemented with 0.5%-0.75% Congo Red. Yeast cells were spotted by 10-fold serial dilutions. Plates were incubated at 30°C and 37°C for 48 h. (C) Bortezomib (BTZ) sensitivity upon autophagy mutants. Fold change in CFU/mL after 48 hours of induction in nitrogen-starvation medium (SD/-N/-AA), with or without BTZ (50 μg/mL). Values shown are means of the experiments. Error bars represent the standard deviation. Unpaired t test: ** (P < 0.05); ns (not significant). All the experiments are n = 2 independent replicates.