Contributions of Ccr4 and Gcn2 to the Translational Response of C. neoformans to Host-Relevant Stressors and Integrated Stress Response Induction

Abstract

In response to the host environment, the human pathogen Cryptococcus neoformans must rapidly reprogram its translatome from one which promotes growth to one which is responsive to host stress. In this study, we investigate the two events which comprise translatome reprogramming: the removal of abundant, pro-growth mRNAs from the translating pool, and the regulated entry of stress-responsive mRNAs into the translating pool. Removal of pro-growth mRNAs from the translating pool is controlled primarily by two regulatory mechanisms, repression of translation initiation via Gcn2, and decay mediated by Ccr4. We determined that translatome reprogramming in response to oxidative stress requires both Gcn2 and Ccr4, whereas the response to temperature requires only Ccr4. Additionally, we assessed ribosome collision in response to host-relevant stress and found that collided ribosomes accumulated during temperature stress but not during oxidative stress. The phosphorylation of eIF2α that occurred as a result of translational stress led us to investigate the induction of the integrated stress response (ISR). We found that eIF2α phosphorylation varied in response to the type and magnitude of stress, yet all tested conditions induced translation of the ISR transcription factor Gcn4. However, Gcn4 translation did not necessarily result in canonical Gcn4-dependent transcription. Finally, we define the ISR regulon in response to oxidative stress. In conclusion, this study begins to reveal the translational regulation in response to host-relevant stressors in an environmental fungus which is capable of adapting to the environment inside the human host. IMPORTANCE Cryptococcus neoformans is a human pathogen capable of causing devastating infections. It must rapidly adapt to changing environments as it leaves its niche in the soil and enters the human lung. Previous work has demonstrated a need to reprogram gene expression at the level of translation to promote stress adaptation. In this work, we investigate the contributions and interplay of the major mechanisms that regulate entry of new mRNAs into the pool (translation initiation) and the clearance of unneeded mRNAs from the pool (mRNA decay). One result of this reprogramming is the induction of the integrated stress response (ISR) regulon. Surprisingly, all stresses tested led to the production of the ISR transcription factor Gcn4, but not necessarily to transcription of ISR target genes. Furthermore, stresses result in differential levels of ribosome collisions, but these are not necessarily predictive of initiation repression as has been suggested in the model yeast.

Keywords: integrated stress response; ribosome collision; stress adaptation; translational control.

FIG 1

Cryptococcus neoformans represses translation in response to temperature and oxidative stresses. (A) A model for stress responsive translatome reprogramming in which Gcn2 and Ccr4 control the entry and exit of mRNAs from the translating pool, respectively. Western blots for phosphorylated eIF2α (B) and northern blots for the RPL2 transcript (C) in C. neoformans in response to a shift to 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT (3-amino-1,2,4-triazole). (D) Polysome profiling of C. neoformans in response to a shift to 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (E) Polysome profiling analysis of samples from panel C after treatment with RNase I.

FIG 2

Gcn2 is primarily required for translational repression under oxidative stress. (A) Serial dilution spot plate analysis of wild-type (WT), gcn2Δ mutant, and GCN2 + gcn2Δ complemented strains. (B) Northern blot analysis for rpl2 transcripts in WT and gcn2Δ mutant strains in response to 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (C) Polysome profiling of the gcn2Δ mutant in response to 30 min at 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (D) Polysome profiling analysis of RNase I-digested samples from panel C.

FIG 3

Ccr4 is required for translational repression in response to temperature and oxidative stresses. (A) Serial dilution spot plate analysis of WT, ccr4Δ mutant, and CCR4 + ccr4Δ complemented strains. Western blots for phosphorylated eIF2α (B) and northern blots for the RPL2 transcript (C) in WT and ccr4Δ strains in response to 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (D) Polysome profiling of the ccr4Δ mutant after 30 min at 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (E) Polysome profiling analysis of RNase I-digested samples from panel D.

FIG 4

Minimal medium is a stressful environment that results in increased repression of translation initiation. (A) Western blots for phosphorylated eIF2α in a WT strain after 30 min of treatment in response to combinations of 30°C and 37°C temperature stress, 0 mM H₂O₂, 0.5 mM H₂O₂, or 1 mM H₂O₂ stress, in either yeast extract-peptone-dextrose (YPD) (complete) or yeast nitrogen base (YNB) + 2% dextrose (YNB-2% Dex) (minimal) medium. (B) Polysome profiling of the conditions in panel A. (C) Serial dilution spot plate analysis of WT, ccr4Δ mutant, and CCR4 + ccr4Δ complemented strains. (D) Western blots for phosphorylated eIF2α in WT and ccr4Δ mutant strains in response to 37°C in nutrient-rich medium (YPD) and minimal medium (YNB-2% Dex). (E) Northern blot analysis for RPL2 transcripts in WT and ccr4Δ mutant strains in response to 37°C in either YPD or YNB-2% dextrose.

FIG 5

Gcn2-mediated translational repression in minimal medium can suppress the mRNA decay defect of ccr4Δ during temperature-responsive translatome reprogramming. (A) Serial dilution spot plate analysis of WT, ccr4Δ, gcn2Δ, and ccr4Δgcn2Δ mutant strains at different temperatures and in the presence of 1 mM H₂O₂ in YPD or YNB-2% dextrose. (B) Northern blot analysis for RPL2 transcripts in WT and ccr4Δ or ccr4Δgcn2Δ strains in response to 37°C in either YPD or YNB-2% dextrose. (C) Polysome profiling of ccr4Δ and ccr4Δgcn2Δ mutants in either YPD or YNB-2% dextrose after 30 min at 37°C.

FIG 6

Temperature and oxidative stresses result in differential integrated stress response (ISR) induction. (A) Western blots for eIF2(alpha) phosphorylation in the WT strain in response to 37°C, 1 mM H₂O₂, 2 mM H₂O₂, and 40 mM 3-AT. (B) Western blots for Gcn4 under the same conditions as panel A. Arrows indicate the band for Gcn4 (C) Northern blot analysis for the ARG1 transcript under the same conditions as for panel A and B. (D) Volcano plots of –log₁₀ P values versus log₂ fold-change for RNA levels in the WT strain (1 mM H₂O₂-treated versus untreated; left), gcn4Δ mutant (1 mM H₂O₂-treated versus untreated; center), and the gcn4Δ mutant and WT (1 mM H₂O₂; right). Genes identified as part of the ISR are shown in green and ribosome biogenesis genes are shown in red. (D) Venn diagram describing the rationale used to determine the stringent ISR regulon. (F) Gene Ontology (GO) enrichment analysis of genes identified as components of the ISR regulon.