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

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.