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Principal Investigator: Xiaorong Lin
Organization: UNIVERSITY OF GEORGIA
Fiscal Year: 2023
Award: $188,750
Funding agency: National Institute of Allergy and Infectious Diseases
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 (≥37C) 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.
Terms: <ACE2><AIDS><Acquired Immune Deficiency><Acquired Immune Deficiency Syndrome><Acquired Immuno-Deficiency Syndrome><Acquired Immunodeficiency Syndrome><Acquired Immunologic Deficiency Syndrome><Air><Antifungal Agents><Antifungal Drug><Antifungal Therapy><Ascomycetes><Ascomycota><Basal Transcription Factor><Basal transcription factor genes><Basidiomycetes><Basidiomycota><Binding><Binding Proteins><Biological Function><Biological Process><C neoformans><C. neoformans><CNS Diseases><CNS disorder><CO2><Candida><Carbon Dioxide><Carbonic Anhydride><Cell Body><Cells><Central Nervous System Diseases><Central Nervous System Disorders><Cerebromeningitis><Cessation of life><Chemicals><Clinical><Complex><Critical Paths><Critical Pathways><Cryptococcosis><Cryptococcus><Cryptococcus neoformans><Cultural Backgrounds><Death><Development><Disease><Disorder><Encephalomeningitis><Exclusion><Future><GeneHomolog><General Transcription Factor Gene><General Transcription Factors><Generalized Growth><Genes><Genetic analyses><Genotoxins><Goals><Growth><High temperature of physical object><Homolog><Homologous Gene><Homologue><Human><Immune Precipitation><Immunoprecipitation><Infection><Intravenous><Investigation><Kinases><Ligand Binding Protein><Ligand Binding Protein Gene><Meningoencephalitis><Messenger RNA><Microbe><Modeling><Modern Man><Molecular><Molecular Interaction><Monilia><Morphogenesis><Mutagens><Names><Non-Polyadenylated RNA><Pathogenesis><Pathogenicity><Pathway interactions><Patients><Phosphorylation><Phosphorylation Site><Phosphotransferase Gene><Phosphotransferases><Physiologic><Physiological><Pilot Projects><Post-Transcriptional Control><Post-Transcriptional Regulation><Process><Protein Binding><Protein Phosphorylation><Proteins><Public Health><Putative RNA-Binding Region><RNA><RNA Binding Domain><RNA Gene Products><RNA Processing><RNA Recognition Motif><RNA Seq><RNA sequencing><RNAseq><RNP Domain><RNP Motif><RNP-1 Signature><Research><Ribonucleic Acid><Saccharomyces><Suppressor Mutations><Temperature><Therapeutic Fungicides><Tissue Growth><Torula><Torulosis><Transcript><Transcription Factor Proto-Oncogene><Transcription factor genes><Translational Inhibition><Translational Repression><Translations><Transphosphorylases><Virulence><Work><Yeast Model System><Yeasts><access to vaccination><access to vaccines><angiotensin converting enzyme 2><angiotensin converting enzyme II><anti-fungal><anti-fungal agents><anti-fungal drug><anti-fungal therapy><antifungals><attenuation><base><bases><bound protein><comparative><developmental><fungal infectious disease treatment><fungal pathogen><fungi pathogen><fungus><genetic analysis><genotoxic agent><high temperature><improved><mRNA><mRNA Translation><morphogenetic process><mortality><mouse model><murine model><mutant><name><named><naming><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><ontogeny><pathogenic fungus><pathway><phospho-proteomics><phosphoproteomics><pilot study><post-transcriptional gene regulation><posttranscriptional><posttranscriptional control><posttranscriptional regulation><prevent><preventing><response><sac fungi><stress granule><trait><transcription factor><transcriptome sequencing><transcriptomic sequencing><translation><vaccination access><vaccination availability><vaccine access><vaccine availability><yeast model>