Calcineurin signaling cascades governing Cryptococcus virulence - Abstract Cryptococcus is one of the most important HIV/AIDS-associated pathogens, causing >220,000 infections, >180,000 deaths, and >15% of all HIV/AIDS-related deaths annually. The ability of C. neoformans to survive at mammalian body temperature and in the presence of other host stress conditions is essential for virulence. We identified the protein phosphatase calcineurin as a major molecular determinant required for Cryptococcus thermotolerance and virulence. In response to temperature stress, calcineurin is activated by Ca2+- calmodulin and acts as a serine/threonine phosphatase. Calcineurin plays broad roles in cryptococcal virulence and is necessary to survive heat, cation, alkaline, and cell wall stress. We have further demonstrated calcineurin plays conserved roles in virulence of other human pathogenic fungi (Candida, Aspergillus, Mucor), and others have shown calcineurin is critical for virulence of Leishmania, Plasmodium, and plant fungal pathogens. However, calcineurin is conserved across eukaryotes, and calcineurin inhibitors are potent immunosuppressants in humans and thus difficult to utilize as antifungal drugs. Studies are in progress developing less immunosuppressive analogs as candidate therapies. Thus, calcineurin is a general, conserved virulence factor in eukaryotic microbial pathogens that can be targeted for therapy, and elucidation of the roles and mechanisms of calcineurin signaling cascades can identify fungal-specific targets and is of general importance. We have achieved several key advances elucidating calcineurin roles in Cryptococcus pathogenesis. We discovered heat and other stresses induce calcineurin to re-localize from the cytoplasm to P-bodies/stress granules, sites of mRNA post-transcriptional/translational control. Via phospho-proteomic studies, we identified calcineurin targets including the transcription factor Crz1 and proteins that localize to P-bodies/stress granules and mediate mRNA processing, stability, and translation. Additionally, we identified stress-response genes regulated by calcineurin-Crz1, as well as mRNAs regulated by calcineurin but not Crz1 through RNA-seq analysis. These findings support our hypothesis that calcineurin controls a bifurcated signaling cascade promoting stress survival and pathogenesis. In one branch, calcineurin stimulates Crz1 nuclear localization and gene expression promoting stress survival and virulence. In a second, less-studied branch, calcineurin undergoes heat-induced re-localization to P-bodies/stress granules and acts on targets governing mRNA processing, stability, and translation, promoting pathogenesis post-transcriptionally. We propose two aims to test this hypothesis. In Aim 1, we will identify, validate, and characterize high temperature specific calcineurin interactors as substrates and effectors via TurboID-proximity labelling and Crz1 ChIP-seq analysis. In Aim 2, we will define the importance of calcineurin re-localization and action on P-body/stress granule targets that enable Cryptococcus to surmount host stress and infect normal and immunocompromised hosts. These studies will elucidate conserved stress-responsive virulence networks of eukaryotic pathogens as therapeutic targets.
