Stress-dependent ribosome-associated proteomics reveals a role for Sis1 in thermotolerance and virulence in Cryptococcus neoformans

ABSTRACT During human infection, Cryptococcus neoformans can disseminate to the central nervous system, causing often-fatal cryptococcal meningitis in individuals with compromised immunity. To acutely adapt to host micro-environments, C. neoformans employs mechanisms to reprogram its translatome to provide cells with tools to survive the stressors of the human host. The identities and functions of ribosome-associated factors that may modulate this process in C. neoformans remain unknown. We hypothesized that following host-related stressors, specific proteins associate with ribosomes and influence stress adaptation in C. neoformans . Utilizing a proteomic approach, termed RiboPROT, we analyzed the protein composition of mRNA-associated ribosomes from cells grown under unstressed (30°C), thermally stressed (shifted to 37°C), and oxidatively stressed (shifted to 2 mM H 2 O 2 ) conditions. We identified unique factors enriched with actively translating ribosomes during host-related stressors. Phenotypic analysis of deletion mutants in selected ribosome-associated factors revealed that the J-domain protein Sis1 is important for growth at elevated temperature, Hsp70 and Hsp90 function, and virulence. The sis1 Δ mutant also exhibited sensitivity to multiple drugs that cause mitochondrial perturbation, and growth on acetate as a sole carbon source suppressed the thermal growth defect at 38°C, suggesting that Sis1 supports mitochondrial homeostasis during thermal stress. Additionally, the sis1 Δ mutant displayed transient hyper-repression of translation initiation during a shift to acute thermal stress at human body temperature but not at human febrile temperature. Global features of stress-responsive translatome reprogramming remained intact in the absence of Sis1, suggesting its role in thermotolerance and virulence is likely linked to regulation of specific targets involved in mitochondrial homeostasis in C. neoformans . IMPORTANCE Fungal pathogens are an underrepresented and emerging global public health threat. In 2022, the World Health Organization recognized Cryptococcus neoformans as the top fungal high-priority pathogen, signifying the urgency for increased research and new therapeutics. The mechanisms by which C. neoformans adapts to the insults of the human body and causes systemic disease remain incompletely understood. Although the ribosome is essential to fungal viability and is targetable by small molecules, the conservation of the eukaryotic ribosome hampers the development of molecules that are selective for the fungal ribosome. Here, we identify stress-dependent ribosome-associated factors in C. neoformans and highlight one identified protein, Sis1, demonstrating its contribution to thermotolerance and virulence. Identification of stress-induced ribosome-associated factors and uncovering their biological role in the cell may identify pathways for selective therapeutic targeting of the fungal ribosome.