A translational checkpoint couples proline sensing to mitochondrial proline catabolism in Candida glabrata

Abstract

Proline catabolism represents a central metabolic and regulatory hub integrating nutrient sensing, stress adaptation, and energy production across diverse organisms. Despite its biological importance, the regulatory mechanisms controlling proline catabolism remain poorly understood in eukaryotic microbes. Here, we describe the transcriptional and translational coordination to catabolize the proline to maintain the cellular homeostasis under stress in the human fungal pathogen Candida glabrata. We identified proline utilisation trigger global translation repression to activates the stress-sensing kinase Gcn2, which phosphorylates eIF2, thereby promoting the activation of the transcription factor Gcn4. Activated Gcn4 upregulates the transcription factor Put3 and the proline transporter Put4. Put3 orchestrates expression of the mitochondrial catabolic enzymes Put1 and Put2, ensuring efficient proline utilization, mitochondrial function, and redox balance. Genetic disruption of PUT3 abolishes proline utilization, impairs mitochondrial function, and severely compromises cellular fitness. Importantly, Put3-mediated proline catabolism is also critical for C. glabrata survival within macrophages and for virulence in systemic infection models. These findings reveal a mechanistic link between proline catabolism, translational regulation, and amino acid sensing in C. glabrata. We propose a regulatory cascade wherein Gcn2-Gcn4-Put3 signaling aligns translational reprogramming with metabolic demands to optimize proline utilization. Thus, this study establishes proline catabolism as a signaling-driven adaptive mechanism essential for fungal metabolism and persistence, rather than merely a nutritional pathway. AUTHOR SUMMARYProline is a versatile amino acid that is essential for cellular metabolism, signaling, stress adaptation, and redox equilibrium. Proline catabolism has been implicated in cancer biology and is increasingly recognized as a key determinant of virulence in diverse pathogens. Although the enzymatic processes of proline use are well characterized, the regulatory mechanisms that sense proline availability and coordinate its metabolic integration remain poorly understood. Here, we identify a hitherto unknown regulatory axis linking proline catabolism to translational reprogramming in Candida glabrata, which seems to be similarly present in numerous fungi. We demonstrate that proline utilization triggers global translational repression via Gcn2-dependent phosphorylation of eIF2, thereby activating the transcription factor Gcn4. Gcn4 is essential for proline utilization, as it controls the expression of PUT3 and PUT4. Notably, PUT3 has no known human counterpart. Our findings establish proline as a metabolic signal that couples translational control to virulence, revealing new opportunities for antifungal intervention.