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.
Highlights
Autophagy is mainly a catabolic process, which is induced by nutrient deficiency and various stress conditions (Yang and Klionsky, 2010)
The genome of C. neoformans strain H99 did not contain orthologs of Atg10, an important and specific E2 enzyme required for Atg12-Atg5 conjugation (Shintani et al, 1999)
The results showed that the deletion of ATG1, ATG13, ATG11, ATG7, ATG5 and ATG12 led to significantly lower autophagic flux, as no green fluorescent protein (GFP) release was detected in the mutant strains after nitrogen starvation for 6 h, whereas significant GFP release was detected in the wild type (WT), indicating that these genes are essential for boosting autophagic flux under nitrogen starvation conditions (Figure 3A)
Summary
Autophagy is mainly a catabolic process, which is induced by nutrient deficiency and various stress conditions (Yang and Klionsky, 2010). The most important and characteristic morphological feature of autophagy is the de novo formation of double-membrane autophagosome, which contains either bulk cytoplasm or selected cargos, depending on the inducing conditions (Inoue and Klionsky, 2010). Formation of PI3P depends on the PI3K complex I, which contains Vps, Vps, Vps30/Atg, Atg and Atg (Petiot et al, 2000; Kihara et al, 2001). Atg is a deconjugation enzyme that liberates Atg from the Atg8-PE complex in the outer membrane of autophagosome after fusion with the vacuole (Kirisako et al, 2000). Atg is the only known integral membrane protein involved in the regulation of autophagosome formation by interacting with the Atg18-Atg complex in a PI3P-dependent manner (Noda et al, 2000; Obara et al, 2008) (Figure 1)
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