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 Ca2+ levels caused by stress, and transduces signals by dephosphorylating protein substrates. Herein, we identified and characterized calcineurin substrates in C. neoformans by employing phosphoproteomic TiO2 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.

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Published Version (Please cite this version)

10.1371/journal.ppat.1005873

Publication Info

Park, Hee-Soo, Eve WL Chow, Ci Fu, Erik J Soderblom, M Arthur Moseley, Joseph Heitman and Maria E Cardenas (2016). Calcineurin Targets Involved in Stress Survival and Fungal Virulence. PLoS Pathog, 12(9). p. e1005873. 10.1371/journal.ppat.1005873 Retrieved from https://hdl.handle.net/10161/13053.

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Scholars@Duke

Soderblom

Erik James Soderblom

Associate Research Professor of Cell Biology

Director, Proteomics and Metabolomics Core Facility

Moseley

Martin Arthur Moseley

Adjunct Professor in the Department of Cell Biology
Heitman

Joseph Heitman

Chair, Department of Molecular Genetics and Microbiology

Joseph Heitman was an undergraduate at the University of Chicago (1980-1984), graduating from the BS-MS program with dual degrees in chemistry and biochemistry with general and special honors. He then matriculated as an MD-PhD student at Cornell and Rockefeller Universities and worked with Peter Model and Norton Zinder on how restriction enzymes recognize specific DNA sequences and how bacteria respond to and repair DNA breaks and nicks. Dr. Heitman moved as an EMBO long-term fellow to the Biocenter in Basel Switzerland where, in studies with Mike Hall and Rao Movva, pioneered the use of yeast as a model for studies of immunosuppressive drug action. Their studies elucidated the central role of FKBP12 in forming complexes with FK506 and rapamycin that inhibit cell signaling and growth, discovered Tor1 and Tor2 as the targets of rapamycin, and contributed to the appreciation that immunosuppressive drugs inhibit signal transduction cascades that are conserved from yeasts to humans.

Dr. Heitman moved to Duke University in 1992, and is a member of the Department of Molecular Genetics and Microbiology where his studies focus on microorganisms addressing fundamental biological questions and unmet medical needs.  Dr. Heitman and colleagues focus on model and pathogenic yeasts including Cryptococcus neoformans and other diverse species from the fungal kingdom. Their studies with fungi as genetic models have revealed biological and genetic principles that can be generalized as models for eukaryotic cell and organism function. These include discovering FKBP12 and Tor1/2 as the targets of the immunosuppressive anti-proliferative natural product rapamycin, elucidating central roles of the calcium activated phosphatase calcineurin governing fungal virulence and morphogenesis and antifungal drug action, deciphering how cells sense and respond to nutrients via permeases, G protein coupled receptors, and the Tor signaling cascade, and illustrating how both model and pathogenic fungi sense both the environment and the infected host. In parallel, their studies address the evolution, structure, and function of fungal mating type loci as models for gene cluster and sex chromosome evolution.  The discovery of an ancestral sex determining locus in the basal fungal lineages involving two HMG domain proteins, SexM and SexP, homologous to the mammalian Sry sex determinant provides insights into both the origins of sex specification and its plasticity throughout the radiation of the fungal and metazoan kingdoms from their last shared common ancestor.  Their discovery of unisexual mating in fungi and subsequent analysis of its impact on the evolution of eukaryotic microbial pathogens provides insights into both microbial evolution and pathogenesis and how sexual reproduction may have first evolved.  Recent studies have unveiled novel mechanisms of antimicrobial drug resistance involving epimutations that silence drug-target genes via RNAi, functions of RNAi in genomic integrity of microbial pathogens, and loss of RNAi in hypervirulent outbreak lineages.

Dr. Heitman is a recipient of the Burroughs Wellcome Scholar Award in Molecular Pathogenic Mycology (1998-2005), the 2002 ASBMB AMGEN award for significant contributions using molecular biology to our understanding of human disease, and the 2003 Squibb Award from the Infectious Diseases Society of America (IDSA) for outstanding contributions to infectious disease research, the 2018 Korsmeyer Award from the American Society for Clinical Investigation, and the 2018 Rhoda Benham Award from the Medical Mycological Society of the Americas.  He is the recipient of an NIH/NIAID MERIT award 2011-2021 in support of studies on fungal unisexual reproduction in microbial pathogen evolution, a Duke University translational research mentoring award in 2012, and a Dean’s Award for Excellence in Mentoring from the Duke Graduate School in 2018.  He has served as an instructor in residence since 1998 for the Molecular Mycology Course at the Marine Biological Laboratory at Woods Hole, MA. Dr. Heitman is an editor for the journals PLOS GeneticsGenetics (2012-2017)PLOS Pathogens (Pearls review editor), Current Genetics (2001-2014)mBio, and Fungal Genetics and Biology; a member of the editorial boards of PLOS BiologyCurrent BiologyCell Host and Microbe, and PeerJ; former editor for PLOS Pathogens (mycology section editor, 2008-2011) and Eukaryotic Cell (2002-2012); an advisory board member for the Fungal Genome Initiative at the Broad Institute, the Fungal Kingdom Genome Project at the Department of Energy Joint Genome Institute, the NIAID Genomic Sequencing Centers for Infectious Diseases, and for the Integrated Microbial Biodiversity Program at the Canadian Institute for Advanced Research (CIFAR); co-chair for the Duke Chancellor’s Science Advisory Council (2009-2010); and co-chair/chair for the FASEB summer conference on Microbial Pathogenesis: Mechanisms of Infectious Disease (2011, 2013).  He was elected a member of the American Society for Clinical Investigation (ASCI) in 2003, a fellow of the Infectious Diseases Society of America (IDSA) in 2003, a fellow of the American Academy of Microbiology in 2004, a fellow of the American Association for the Advancement of Science (AAAS) in 2004, a member of the Association of American Physicians (AAP) in 2006, and a member of the American Academy of Arts & Sciences in 2020.  Dr. Heitman was an investigator with the Howard Hughes Medical Institute from 1992 to 2005. Dr. Heitman served as the director for the Duke University Program in Genetics and Genomics (UPGG) from 2002-2009 (including writing two funded competitive renewals for the T32 NIH training grant and establishing the annual program retreat). He was the founding director for the Center for Microbial Pathogenesis (now called the Center for Host-Microbial Interactions, CHoMI) and served in this capacity January 2002-October 2014.  He is currently the director of the Tri-institutional (Duke, UNC-CH, NC State) Molecular Mycology and Pathogenesis Training Program (MMPTP) (since July 1, 2012), and Chair of the Department of Molecular Genetics and Microbiology (since September 1, 2009).


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