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. OPEN ACCESS Citation: Roberto TN, Lima RF, Pascon RC, Idnurm A, Vallim MA (2020) Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans. PLoS ONE 15(4): e0230981. https://doi.org/10.Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files.individuals [2]. The therapy against cryptococcosis requires a long period of treatment with antifungal drugs used singly or in combination, some of which are highly toxic to the patient [3]. Due to the long period of clinical treatment, some reports indicate that fungi of the genus Cryptococcus may have a high potential to antifungal resistance, which could explain the therapeutic failures and recurrent relapses in the patients with cryptococcosis [4]. Thus, further studies for new strategies that contribute with the knowledge about the treatment of this mycosis, leading to the deficiency of growth, multiplication, and/or survival of the fungus in the host are of great importance. In this context, the elucidation of autophagic pathways in fungi may provide new insights into the relationship established during the infection process between pathogen and host [5].Autophagy is an intracellular mechanism responsible for degradation and recycling of macr...

Autophagy Regulates Fungal Virulence and Sexual Reproduction in Cryptococcus neoformans

“…To investigate the role of the core autophagy machinery genes (ATGs), we knocked out all 14 ATG genes in C. neoformans. Each single deletion mutant showed the expected autophagy phenotype, such as impaired growth and survival upon nitrogen starvation, as previously reported by other groups (Hu et al, 2008;Oliveira et al, 2016;Gontijo et al, 2017;Ding et al, 2018;Zhao et al, 2019a;Roberto et al, 2020). However, our results suggested that autophagy-related genes may also be involved in other cellular functions, such as maintaining the integrity of cell membranes.…”

Transcriptomic and Ultrastructural Analyses of Pyricularia Oryzae Treated With Fungicidal Peptaibol Analogs of Trichoderma Trichogin

“…Besides, we found many genes involved in DNA repair, remodeling, and maintenance of chromatin structure as differentially expressed, thus suggesting that DNA damage was likely occurring in fungal cells, probably contributing to cell death. Whether the positively charged peptide directly causes DNA damage or the damage is rather an indirect effect of ROS accumulation, which at high levels is lethal for fungal cells ( Roberto et al, 2020 ) needs to be further investigated.…”

“…More than 40 autophagy-related (ATG) genes have been identified in fungi ( Roberto et al, 2020 ). Our transcriptomic analysis found that ATG3, ATG4, ATG7, ATG9 and ATG17 encoding genes were upregulated.…”

“…Our transcriptomic analysis found that ATG3, ATG4, ATG7, ATG9 and ATG17 encoding genes were upregulated. In particular, ATG4 encodes a cysteine protease necessary for processing ATG8, an essential gene for autophagosome synthesis and thus for autophagy pathway activation ( Liu et al, 2010 ; Ren et al, 2018 ; Roberto et al, 2020 ). Besides, in P. oryzae , ATG3 and ATG7 putatively play a role in phagophore and autophagosome expansion, ATG9 in recycling, and ATG17 in the initiation of autophagy ( Kershaw and Talbot, 2009 ).…”

Current Perspectives on Uniparental Mitochondrial Inheritance in Cryptococcus neoformans

“…Autophagy does not play a role in mitochondrial inheritance in C. neoformans either. Nishimura et al have shown that disruption of ATG8 (responsible for the creation of the autophagosome vesicle membrane [ 38 ] and NUC1 (an endonuclease involved in mtDNA degradation) do not have an effect on the preferential inheritance of MAT a mtDNA inheritance in Cryptococcus [ 24 ] ( Table 1 ). Consistently, we found that a unilateral cross of wild type with either atg3 Δ or aif1 Δ mutant defective in autophagy-related E2-like conjugation enzyme and mitochondrial nuclease respectively yielded the same UMI pattern as a cross between two wild type partners.…”