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. 2019 Nov 26:10:2690.
doi: 10.3389/fmicb.2019.02690. eCollection 2019.

Conserved Autophagy Pathway Contributes to Stress Tolerance and Virulence and Differentially Controls Autophagic Flux Upon Nutrient Starvation in Cryptococcus neoformans

Xueru Zhao  1 , Weijia Feng  1 , Xiangyang Zhu  1 , Chenxi Li  2 , Xiaoyu Ma  2 , Xin Li  1 , Xudong Zhu  2 , Dongsheng Wei  1
Affiliations

Conserved Autophagy Pathway Contributes to Stress Tolerance and Virulence and Differentially Controls Autophagic Flux Upon Nutrient Starvation in Cryptococcus neoformans

Xueru Zhao et al. Front Microbiol. .

Abstract

Autophagy is mainly a catabolic process, which is used to cope with nutrient deficiency and various stress conditions. Human environment often imposes various stresses on Cryptococcus neoformans, a major fungal pathogen of immunocompromised individuals; therefore, autophagic response of C. neoformans to these stresses often determines its survival in the host. However, a systematic study on how autophagy related (ATG) genes influence on autophagic flux, virulence, stress response and pathogenicity of C. neoformans is lacking. In this study, 22 ATG-deficient strains were constructed to investigate their roles in virulence, pathogenesis, stress response, starvation tolerance and autophagic flux in C. neoformans. Our results showed that Atg6 and Atg14-03 significantly affect the growth of C. neoformans at 37°C and laccase production. Additionally, atg2Δ and atg6Δ strains were sensitive to oxidative stress caused by hydrogen peroxide. Approximately half of the atgΔ strains displayed higher sensitivity to 1.5 M NaCl and remarkably lower virulence in the Galleria mellonella model than the wild type. Autophagic flux in C. neoformans was dependent on the Atg1-Atg13, Atg5-Atg12-Atg16, and Atg2-Atg18 complexes and Atg11. Cleavage of the green fluorescent protein (GFP) from Atg8 was difficult to detect in these autophagy defective mutants; however, it was detected in the atg3Δ, atg4Δ, atg6Δ and atg14Δ strains. Additionally, no homologs of Saccharomyces cerevisiae ATG10 were detected in C. neoformans. Our results indicate that these ATG genes contribute differentially to carbon and nitrogen starvation tolerance in C. neoformans compared with S. cerevisiae. Overall, this study advances our knowledge of the specific roles of ATG genes in C. neoformans.

Keywords: ATG genes; Atg8; Cryptococcus neoformans; autophagy; starvation; stress tolerance; virulence.

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Figures

FIGURE 1
FIGURE 1
Overview of the autophagy pathway in yeast. Five key stages of autophagy are as follows: (1) Phagophore nucleates at the PAS and sequesters vesicle formation in yeast; (2) phagophore expands to sequester the cargo; (3) phagophore closes the double-membrane autophagosomes; (4) autophagosome fuses with the vacuole, releasing the inner vesicle, known as the autophagic body; (5) autophagic bodies are degraded by vacuolar hydrolases, and the products are released into the cytosol by various permeases. Revised from Nakatogawa et al., 2009.
FIGURE 2
FIGURE 2
Two Atg14 homologous proteins in Cryptococcus neoformans. Putative conserved domains of Atg14-03 (CNAG_03608) and Atg14-05 (CNAG_05500), and amino acid sequence alignments of two Atg14 homologs are shown.
FIGURE 3
FIGURE 3
Nitrogen or carbon starvation induces autophagy in C. neoformans. (A,B) Analysis of GFP-Atg8 processing by western blotting. H99 and atgΔ strains transformed with a plasmid expressing the EGFP-Atg8 fusion protein were collected after shifting to nitrogen starvation medium (SD-N) (A) or carbon starvation medium (SD-C) (B) at the indicated times. Total proteins were extracted and subjected to immunoblot analysis; β-actin was used as the loading control. (C) Localization of EGFP-Atg8 upon nutrient starvation. H99, atg1Δ and atg13Δ cells in log phase (starved; 0 h) or after shifting to SD-N or SD-C medium containing 10 μg/mL nocodazole for 4 h were observed by both fluorescence microscopy and differential interferential contrast (DIC) with a laser scanning confocal microscope. (D) Autophagic body formation. H99 and atgΔ strains were incubated in SD-N or SD-C medium containing 1 mM PMSF and 10 μg/mL nocodazole for 4 h and subjected to DIC microscopy. Arrows indicate autophagic bodies within vacuoles.
FIGURE 4
FIGURE 4
Phenotypes of atgΔ mutants of C. neoformans. (A) Thermotolerance test. Serial 10-fold dilutions of H99 and atgΔ strains were spotted onto YPD medium and incubated at 37°C. (B) Laccase activity assay to determine melanin production. Each strain was spotted and cultured on Asn medium containing 0.1% glucose and 100 mg/L norepinephrine at 30°C for 3 days. (C) Relative laccase activity of H99, atg2Δ and atg6Δ strains using ABTS as a substrate. The assay was performed three times. Asterisks indicate significant differences between the wild-type (H99) and mutant strains (∗P < 0.05; ∗∗∗P < 0.001; Student’s t-test). (D) Qualitative and quantitative analysis of LAC1 expression in atg2Δ and atg6Δ by reverse transcription PCR (RT-PCR) and quantitative real-time (qRT-PCR) upon induction by 0.1% glucose for 5 h. The assay was performed three times. Asterisks indicate significant differences (∗∗∗P < 0.001; ****P < 0.0001; Student’s t-test). (E,F) Sensitivity of atgΔ to NaCl and H2O2. Serial 10-fold dilutions of H99 and atgΔ strains were spotted on agar plates containing 1.5 M NaCl or 10 mM H2O2 and incubated at 30°C.
FIGURE 5
FIGURE 5
Tolerance of atgΔ strains to nitrogen and/or carbon starvation. (A–C) H99 and atgΔ strains were grown in liquid YPD for 16 h and then grown on SD-N medium (A), SD-C medium (B) or PBS (C) for the indicated durations. Dilutions were grown on YPD plates for 2 days.
FIGURE 6
FIGURE 6
Role of ATG genes in the virulence of C. neoformans. Survival of Galleria mellonella larvae infected with 106 cells of H99 or atgΔ strains. The larvae were incubated at 30°C, and their survival was monitored daily.
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