Calcineurin Targets Involved in Stress Survival and Fungal Virulence

Abstract

Calcineurin governs stress survival, sexual differentiation, and virulence of the human fungal pathogen Cryptococcus neoformans. Calcineurin is activated by increased Ca ²⁺ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO ₂ enrichment and quantitative mass spectrometry. The identified targets include the transactivator Crz1 as well as novel substrates whose functions are linked to P-bodies/stress granules (PBs/SGs) and mRNA translation and decay, such as Pbp1 and Puf4. We show that Crz1 is a bona fide calcineurin substrate, and Crz1 localization and transcriptional activity are controlled by calcineurin. We previously demonstrated that thermal and other stresses trigger calcineurin localization to PBs/SGs. Several calcineurin targets localized to PBs/SGs, including Puf4 and Pbp1, contribute to stress resistance and virulence individually or in conjunction with Crz1. Moreover, Pbp1 is also required for sexual development. Genetic epistasis analysis revealed that Crz1 and the novel targets Lhp1, Puf4, and Pbp1 function in a branched calcineurin pathway that orchestrates stress survival and virulence. These findings support a model whereby calcineurin controls stress and virulence, at the transcriptional level via Crz1, and post-transcriptionally by localizing to PBs/SGs and acting on targets involved in mRNA metabolism. The calcineurin targets identified in this study share little overlap with known calcineurin substrates, with the exception of Crz1. In particular, the mRNA binding proteins and PBs/SGs residents comprise a cohort of novel calcineurin targets that have not been previously linked to calcineurin in mammals or in Saccharomyces cerevisiae. This study suggests either extensive evolutionary rewiring of the calcineurin pathway, or alternatively that these novel calcineurin targets have yet to be characterized as calcineurin targets in other organisms. These findings further highlight C. neoformans as an outstanding model to define calcineurin-responsive virulence networks as targets for antifungal therapy.

Author Summary

Calcineurin is a Ca ²⁺/calmodulin-dependent protein phosphatase essential for stress survival, sexual development, and virulence of the human fungal pathogen Cryptococcus neoformans and other major pathogenic fungi of global human health relevance. However, no calcineurin substrates are known in pathogenic fungi. Employing state-of-the-art phosphoproteomic approaches we identified calcineurin substrates, including calcineurin itself and the conserved Crz1 transcriptional activator known to function in calcium signaling and stress survival. Remarkably, our study also identified novel calcineurin targets involved in RNA processing, stability, and translation, which colocalize together with calcineurin in stress granules/P-bodies upon thermal stress. These findings support a model whereby calcineurin functions in a branched pathway, via Crz1 and several of the identified novel targets, that governs transcriptional and posttranscriptional circuits to drive stress survival, sexual development, and fungal virulence. Our study underscores C. neoformans as an experimental model to define basic paradigms of calcineurin signaling in global thermostress responsive virulence networks that can be targeted for fungal therapy.