Ribosome Heterogeneity in Cryptococcus neoformans

Invasive fungal infections are a major cause of co-morbidity and mortality in patients living with immunodeficiency and transplantation. A limited antifungal arsenal and emergence of antifungal drug resistance have made the need for novel antifungal therapies paramount. Cellular protein synthesis carried out by the ribosome is a known and well-vetted target for pharmaceutical intervention in prokaryotes and eukaryotes, but has not been exploited in antifungal development because of the presumed inability to target the core catalytic function of the ribosome with specificity in fungi. Using the human pathogen Cryptococcus neoformans, we have leveraged two approaches to identify factors that associate with the fungal ribosome under stress conditions encountered in the host. Using a homology based approach, we have identified Cryptococcus-specific homologues of the conserved eukaryotic mRNA cap-binding complex (eIF4F) that we have names alternative eIF4G (AFG1) and alternative eIF4E (AFE1). Deletion of AFG1 results impairs hypoxia adaptation, which is a major pathogenesis-associated pathway in the major human fungal pathogens. Further work on the function of Afg1 and Afe1 as components of a novel eIF4F complex are outlined in Aim 1, and the identification of the mRNA targets they bind is outlined in Aim 3. Our second approach developed a novel proteomic pipeline that we’ve termed RiboPROT to identify the factors associating with translating ribosomes during adaptation to temperature stress and oxidative stress. Aim 2 of this proposal will investigate the role of these proteins in stress-responsive translation and pathogenesis. We have prioritized hits from our initial RiboPROT experiments to investigate further, and propose to identify additional factors under additional conditions relevant to cryptococcal pathogenesis and biology. For those with RNA-binding domains, cognate mRNA targets will be identified in Aim 3, identifying stress responsive translational regulons. At the conclusion of these studies, we will have a comprehensive set of ribosome-associating factors to investigate in future work as antifungal targets. These unique stress-specific and fungi-specific ribosome-associating factors may provide a novel pathway to targeting the fungal ribosome with specificity.