Dataset Information

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

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, atg2? and atg6? strains were sensitive to oxidative stress caused by hydrogen peroxide. Approximately half of the atg? 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 atg3?, atg4?, atg6? and atg14? 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.

SUBMITTER: Zhao X

PROVIDER: S-EPMC6988817 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Zhao Xueru X Feng Weijia W Zhu Xiangyang X Li Chenxi C Ma Xiaoyu X Li Xin X Zhu Xudong X Wei Dongsheng D

Frontiers in microbiology 20191126

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 res ...[more]

PMID: 32038502

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Project description:MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism.Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks' balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.

Dataset Information

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

Publications

Conserved Autophagy Pathway Contributes to Stress Tolerance and Virulence and Differentially Controls Autophagic Flux Upon Nutrient Starvation in <i>Cryptococcus neoformans</i>.

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