Define the molecular bases for cryptococcal adaptation to host conditions by the RAM pathway

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Abstract Cryptococcal meningoencephalitis is responsible for 15% of the total deaths of AIDS patients. There is no vaccine available for cryptococcosis and the disease claims hundreds of thousands of lives each year, with the global mortality rates of ~70% despite current antifungal therapies. The challenges of preventing and treating this disease motivate the investigation of cryptococcal pathways that are critical for pathogenesis. As Cryptococcus neoformans is an environmental basidiomycetous yeast, the ability to survive and amplify in conditions physiologically relevant for the human host is a prerequisite for its pathogenesis. Both high temperatures (≥37C) and CO2 levels (≥5%) in humans differ considerably from the fungus' primary environmental niches (CO2 in ambient air is ~0.04%). Accordingly, environmental cryptococcal isolates that are CO2-sensitive showed drastic virulence attenuation in mouse models of cryptococcosis. The applicant found that disruption of any of the conserved components of the RAM pathway in the CO2-tolerant clinical isolate H99, including the effector kinase Cbk1, rendered the strain unable to grow at host temperatures or CO2 levels. Consistently, the cbk1 mutant is avirulent in both an intranasal infection model and an intravenous infection model of cryptococcosis. Thus, understanding how the RAM pathway regulates cryptococcal thermotolerance and CO2 tolerance will inform us about how this fungus adapts to host conditions to cause diseases in humans. As nothing is known about downstream effectors of the RAM pathway in basidiomycete fungi, the applicant carried out a pilot suppressor screen of the cryptococcal cbk1 mutant. Analyzing the suppressor mutants revealed that disruption of either of the two RNA-processing regulators, Ssd1 and Psc1, partially restored cbk1's thermotolerance and CO2 tolerance. Ssd1 is conserved among fungi and a known target of Cbk1 based on studies in ascomycetes, including model yeasts or pathogenic Candida species. In the absence of phosphorylation by Cbk1, Saccharomyces Ssd1 translocates to processing or P-bodies and stress granules, thereby suppressing translation of its bound mRNAs. By contrast, Psc1 is an uncharacterized protein with a PARN RNA-recognition motif. The PARN motif is present in many eukaryotic lineages including basidiomycete fungi and humans, but surprisingly absent in ascomycetes such as Saccharomyces or Candida species. Based on these observations, the applicant hypothesizes that thermotolerance and CO2 tolerance in Cryptococcus are regulated by the RAM pathway  at least partly  at the post-transcriptional level. In this exploratory R21 application, the applicant seeks to (1) define Cbk1 kinase downstream targets in Cryptococcus by comparative phosphoproteomics and extensive suppressor screens and (2) identify biological processes controlled by the RAM pathway by defining the proteins and mRNAs bound by Ssd1 and Psc1 in the presence and absence of Cbk1. These findings will deepen our understanding of how C. neoformans adapts to host conditions and may reveal candidates that can be exploited for novel targets against this deadly disease.