Abstract: The pathogenic fungus Cryptococcus neoformans continues to be the cause of 15% of AIDS-related mortality, primarily in under-resourced areas of the globe. The work outlined in the proposal is a continuation of work investigating post-transcriptional and translational mechanisms of stress adaptation in this important fungal pathogen and investigate their conservation across clinical isolates from AIDS-associated cryptococcosis in Africa. We have implicated translatome reprogramming as a requirement for the adaptation to host- temperature stress as well as oxidative stress. This reprogramming is regulated at two points: the entry of mRNAs into the translating pool through regulation of translation initiation, and the removal of mRNAs from the pool by deadenylation-dependent mRNA decay. These two pathways cooperate in a stress-specific and intensity-dependent manner to achieve a stress-adaptive translatome. In this proposal, we propose three specific aims that outline a mechanistic investigation of these processes. First, we will investigate the translational regulation of Gcn4, the transcription factor that controls expression of the integrated stress response (ISR) regulon. We will define the C. neoformans ISR, and define the direct targets of Gcn4 in C. neoformans. Second, we will build on preliminary data demonstrating that translation initiation regulation by the kinase Gcn2 is activated by perturbations in other post-transcriptional and translation regulatory pathways suggesting a role for Gcn2 as a failsafe responder to stress. We will investigate the contributions to translatome reprogramming of these additional pathways, Hog1/p38, Nonsense Mediate Decay (NMD) and Ribosome Quality Control (RQC) and investigate the role of Gcn2 activation in either suppressing or exacerbating defects in translatome reprogramming or virulence phenotype. Finally, we will investigate the mechanisms of translatome reprogramming in a panel of environmental C. neoformans isolates, as well as a large collection of clinical isolates of C. neoformans from people with AIDS-associated cryptococcosis in Africa that have accompanying outcome data and are fully genome sequenced. We will investigate the thresholds for activation of these pathways and the intensity and duration of responses across the isolates, assessing temperature dependent changes in translational output and both cytoplasmic and mitochondrial intracellular reactive oxygen species as ISR activators. We will also determine if the ISR regulon differs across the panel of clinical isolates. This work will define pathways responsible for the agility of this important pathogen that differ from non-pathogen environmental relatives and from the model yeast and identify additional targets for future therapeutic interventions.