A PAS Protein Directs Metabolic Reprogramming during Cryptococcal Adaptation to Hypoxia

Abstract

To aerobic organisms, low oxygen tension (hypoxia) presents a physiological challenge. To cope with such a challenge, metabolic pathways such as those used in energy production have to be adjusted. Many of such metabolic changes are orchestrated by the conserved hypoxia-inducible factors (HIFs) in higher eukaryotes. However, there are no HIF homologs in fungi or protists, and not much is known about conductors that direct hypoxic adaptation in lower eukaryotes. Here, we discovered that the transcription factor Pas2 controls the transcript levels of metabolic genes and consequently rewires metabolism for hypoxia adaptation in the human fungal pathogen Cryptococcus neoformans Through genetic, proteomic, and biochemical analyses, we demonstrated that Pas2 directly interacts with another transcription factor, Rds2, in regulating cryptococcal hypoxic adaptation. The Pas2/Rds2 complex represents the key transcription regulator of metabolic flexibility. Its regulation of metabolism rewiring between respiration and fermentation is critical to our understanding of the cryptococcal response to low levels of oxygen.IMPORTANCEC. neoformans is the main causative agent of fungal meningitis that is responsible for about 15% of all HIV-related deaths. Although an obligate aerobic fungus, C. neoformans is well adapted to hypoxia conditions that the fungus could encounter in the host or the environment. The sterol regulatory element binding protein (SREBP) is well known for its role in cryptococcal adaptation to hypoxia through its regulation of ergosterol and lipid biosynthesis. The regulation of metabolic reprogramming under hypoxia, however, is largely unknown. Here, we discovered one key regulator, Pas2, that mediates the metabolic response to hypoxia together with another transcription factor, Rds2, in C. neoformans The findings help define the molecular mechanisms underpinning hypoxia adaptation in this and other lower eukaryotes.

Keywords: Snf1; Sre1; carbon metabolism; ergosterol; hypoxia; metabolism; obligate aerobe; transcription factors.

FIG 1

C. neoformans cells reprogram central carbon metabolism in response to hypoxia. (A) GOCircle of significantly changed functional categories of DEGs of wild-type cells under hypoxia relative to normoxia conditions. The dots indicate the upregulated and downregulated genes within a specific GO category. The gray bars in the center indicate the P values of GO terms, with higher bars representing higher significance of the GO term category. The description of GO categories is listed in the table at the bottom. (B) Heat map of the DEGs within the significantly changed GO categories under hypoxia relative to normoxia conditions. FPKM, fragments per kilobase per million. (C) DEGs that are involved in central metabolism pathways in response to hypoxia. See the details about the DEGs in Data Set S1 to Data Set S3 in the supplemental material. Red dots represent the number of carbon atoms. G6P, glucose-6-phosphate; F6P, fructose-6-phosphate. AKG, alpha-ketoglutarate; GLU, glutamic acid.

FIG 2

Pas2 regulates hypoxic growth in an SREBP-independent manner in C. neoformans. (A) Hypoxic growth of the nine PAS gene deletion mutants. Serial dilutions of cultures grown overnight were spotted onto YPD medium and cultured in a hypoxia chamber with 0.1% oxygen and 5% carbon dioxide at 37°C. Cells cultured in an incubator with 5% carbon dioxide at 37°C were set as the normoxia controls. Pictures were taken after 2 days of incubation with a fabric background. (B) Epistasis assay between PAS2 and SRE1 under hypoxia. (C) Epistasis assay between PAS2 and STP1 under hypoxia. Cells in panels B and C were cultured in the same way as described above for panel A.

FIG 3

Deletion of PAS2 abolishes the induction of metabolic genes in response to hypoxia in C. neoformans. (A) Heat map of the DEGs in comparison of the pas2Δ mutant and the WT under hypoxia. (B) GOCircle of significantly changed functional categories of DEGs in the pas2Δ mutant compared to the WT under hypoxia. The dots indicate the upregulated and downregulated genes within a specific GO category. The description of GO categories is listed in the table (Data Set S5). (C) Volcano plot of DEGs in the WT under hypoxia compared to normoxia. The colored dots indicate the DEGs that cannot be induced in the pas2Δ mutant under hypoxia. The gene identifications of DEGs that are involved in metabolism as shown in Fig. 1C are shown in the plot. (D) Volcano plot of DEGs in the pas2Δ mutant compared to the WT under hypoxia.

FIG 4

Both the PAS and zinc finger domains are required for the function of Pas2 in regulating hypoxia growth in C. neoformans. (A) Diagram of the Pas2 protein and the conserved sequence alignment of its PAS and zinc finger (ZF) domains. (B) Fluorescence of mCherry-tagged alleles of Pas2. Cells were cultured in liquid YPD medium at 30°C under ambient air overnight. Hoechst staining was used to visualize nuclei. DIC, differential interference contrast. (C) Assay of hypoxic growth of the pas2Δ mutant transformed with different alleles of PAS2.

FIG 5

Pas2 interacts with Rds2 in regulating hypoxic adaptation in C. neoformans. (A) CoIP/MS hits of potential interacting partners of Pas2 under normoxia and hypoxia. Pas2 itself is highlighted in yellow, and three transcription factors are highlighted in purple. (B) Information on proteins from the CoIP/MS hits. (C) Hypoxic growth of three transcription factor mutants identified by CoIP/MS. (D) Fluorescence colocalization of Pas2 and Rds2. Rds2 and Pas2 were tagged with mNeonGreen and mCherry, respectively. Cells were cultured in liquid YPD medium at 30°C under ambient air overnight. DAPI staining was used to visualize nuclei. (E) Validation of the interaction between Pas2 and Rds2 by CoIP/WB. A Pas2-mCherry- and Rds2-6×FLAG-coexpressing strain was cultured under both normoxia and hypoxia conditions. Cells were fixed and lyophilized for total protein extraction. RFP-trap was used for pulldown, and anti-FLAG antibody was used in the subsequent WB assay. An Rds2-mNeonGreen- and Pas2-2×FLAG-coexpressing strain was used for the reciprocal CoIP/WB assay, in which mNeonGreen-trap was used for pulldown and anti-FLAG antibody was used for the subsequent WB assay. The strains expressing only FLAG-tagged Rds2 or Pas2 were included as negative controls. (F) Epistasis assay between PAS2 and RDS2.

FIG 6

Pas2/Rds2 mediates metabolic reprogramming under hypoxia in C. neoformans. (A) Pas2 and Rds2 regulate hypoxic growth in addition to alternative carbon source utilization. The medium with 1% yeast extract and 2% peptone was used as the base medium. Two percent glucose, glycerol, ethanol, or sodium acetate was supplemented into the base medium. To test carbon source utilization, a spotting assay with serial dilutions was conducted under normoxia conditions. Hypoxic growth of the wild-type strain and mutants was tested on YPD medium. (B) Proposed model of Pas2/Rds2-mediated metabolic reprogramming under hypoxia in C. neoformans. Under normoxia, TCA cycle-coupled OXOPHOS is the main energy source to fuel the biological processes in aerobic cells. Under hypoxia, cells reprogram metabolism to avoid the accumulation of reactive oxygen species (ROS) due to the lack of oxygen as the final electron acceptor. The energy supply changes from oxygen-dependent OXOPHOS to glycolytic fermentation. Meanwhile, cells reshuffle the TCA cycle to avoid producing excessive reducing agents and subsequently prevent the accumulation of harmful ROS. Alternative metabolic pathways, including glyoxylate shunt and gluconeogenesis pathways, are upregulated to balance the redox homeostasis and compensate for the lack of building blocks. In addition, cells may reverse the TCA cycle into a reductive direction to maintain the carbon supply for acetyl-CoA, citrate, and fatty acids from glutamine in hypoxia. PEP, phosphoenolpyruvate; FAD, flavin adenine dinucleotide; FADH₂, reduced flavin adenine dinucleotide.