Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation.
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2014-04
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Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.
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Janbon, G, KL Ormerod, D Paulet, EJ Byrnes, V Yadav, G Chatterjee, N Mullapudi, C Hon, et al. (2014). Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation. PLoS Genet, 10(4). p. e1004261. 10.1371/journal.pgen.1004261 Retrieved from https://hdl.handle.net/10161/8468.
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Scholars@Duke
Vikas Yadav
John Robert Perfect
Research in my laboratory focuses around several aspects of medical mycology. We are investigating antifungal agents (new and old) in animal models of candida and cryptococcal infections. We have examined clinical correlation of in vitro antifungal susceptibility testing and with in vivo outcome. Our basic science project examines the molecular pathogenesis of cryptococcal infections. We have developed a molecular foundation for C. neoformans, including transformation systems, gene disruptions, differential gene expression screens, and cloning pathogenesis genes. The goal of this work is to use C. neoformans as a model yeast system to identify molecular targets for antifungal drug development. There are a series of clinical trials in fungal infections which are being coordinated through this laboratory and my work also includes a series of antibiotic trials in various aspects of infections. Finally, we have now been awarded a NIH sponsored Mycology Unit for 5 years with 6 senior investigators which is focused on C. neoformans as a pathogenic model system, but will include multiple areas of medical mycology from diagnosis to treatment.
Jennifer Lodge
Jennifer Lodge, Ph.D., a professor of molecular genetics and microbiology, is Duke’s vice president for Research & Innovation.
As the university’s chief research and innovation officer, Lodge leads oversight of Duke’s $1.2 billion annual research portfolio, including grants administration, ethical practices and commercialization. Lodge works with campus and medical center research staff, faculty and trainees, and is a key figure in Duke’s connection with external partners.
Before coming to Duke in January 2022, Lodge served as vice chancellor for research and senior associate dean for research for the School of Medicine at Washington University in St. Louis (WUSTL). There, she was responsible for WUSTL’s research development, ethics, education, compliance and research administration systems, and earned a reputation for encouraging innovation and entrepreneurship.
Lodge’s own research is on the human pathogenic fungus Cryptococcus neoformans. Her lab has been funded continuously by NIH for more than two decades, with as many as three prestigious R01 grants at one time. Her lab in the Duke University School of Medicine continues to explore the biochemical processes by which this fungus builds its cell walls. Such knowledge could lead to new antifungal therapies and vaccines.
Lodge is a fellow of the American Academy of Microbiology, the American Association for the Advancement of Science (AAAS) and the National Academy of Inventors. She is also the former chair of the Group on Research at the American Association of Medical Colleges (AAMC).
Joseph Heitman
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 Genetics, Genetics (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 Biology, Current Biology, Cell 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).
Fred Samuel Dietrich
My laboratory is interested in fungal genomics.
In particular we use genomic sequencing of fungal strains and species in comparative analysis. Starting with the sequencing of Saccharomyces cerevisiae strain S288C, I have been involved in the genome sequencing and annotation of Ashbya gossypii, Cryptococcus neoformans var. grubii and ~100 additional S. cerevisiae strains. We currently use Illumina paired end and mate paired sequencing, as this is at presently the most cost effective widely used technology capable of generating high accuracy, zero gap whole genome sequences. The 100-genomes S. cerevisiae data as well as the fully updated fully annotated A. gossypii sequence (Genbank numbers AE016814-AE016820), which spans all seven chromosomes from telomere to telomere, were generated using Illumina data. In my laboratory we strive to utilize comparative genomics data to understand aspects of basic fungal biology. Some of our specific areas of interest are filamentous growth, mapping of complex traits, horizontal gene transfer, and identification of RNA coding genes. This work involves a combination of experimental work and bioinformatics analysis. Research in S. cerevisiae has greatly benefitted from an accurate, annotated S. cerevisiae reference genome, and that research into the tremendous diversity in this organism will similarly benefit from the availability of a large number of accurate, fully annotated genome sequences. The use of genomic information to better understand the biology of these organisms, and this is what students in my laboratory generally work on.
What is the set of genes found in a pathogenic fungus such as Cryptococcus?
Our interest in this human pathogen is to expand beyond looking at one isolate and to investigate the diversity in the population. Are there genes found in some Cryptococcus neoformans isolates but not in others? Are there regions of the genome or individual genes which are highly diverged between Cryptococcus isolates? Efforts are now underway at Stanford University to sequence the genome of the JEC21 strain of Cryptococcus. This is a strain that has been agreed upon by the community of Cryptococcus researchers as a reference strain. Obtaining the DNA sequence of this strain is only the start however. From that sequence identifying the complete set of genes will be a considerable challenge requiring both bioinformatic as well as experimental tools. While this work on gene identification is going on we plan on addressing the question of how much do other Cryptococcus isolates differ from JEC21.
What is the set of genes in humans?
The complete DNA sequence of human and mouse will become available soon. This does not mean that we will know the complete set of human or mouse genes. Our current state of knowledge does not allow us to accurately predict human genes directly from DNA sequence. We are interested in applying to the human genome some of the experimental and bioinformatic tools we are developing and utilizing in fungal systems.
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