57 results on '"Christina A, Cuomo"'
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2. More evidence of Impact Factor Mania
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Patrick D. Schloss and Christina A. Cuomo
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Microbiology ,QR1-502 - Published
- 2023
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3. 2023 Acknowledgment of Microbiology Spectrum Ad Hoc Reviewers
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Christina A. Cuomo
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Microbiology ,QR1-502 - Published
- 2023
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4. Opening Up Editorial Boards
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Christina A. Cuomo
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editorial board ,open application ,Microbiology ,QR1-502 - Published
- 2023
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5. 2022 Acknowledgment of Microbiology Spectrum Ad Hoc Reviewers
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Christina A. Cuomo
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Microbiology ,QR1-502 - Published
- 2022
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6. Genomic Variation across a Clinical Cryptococcus Population Linked to Disease Outcome
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Poppy Sephton-Clark, Jennifer L. Tenor, Dena L. Toffaletti, Nancy Meyers, Charles Giamberardino, Síle F. Molloy, Julia R. Palmucci, Adrienne Chan, Tarsizio Chikaonda, Robert Heyderman, Mina Hosseinipour, Newton Kalata, Cecilia Kanyama, Christopher Kukacha, Duncan Lupiya, Henry C. Mwandumba, Thomas Harrison, Tihana Bicanic, John R. Perfect, and Christina A. Cuomo
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Cryptococcus ,GWAS ,aneuploidy ,genome sequencing ,sugar transporters ,virulence ,Microbiology ,QR1-502 - Abstract
ABSTRACT Cryptococcus neoformans is the causative agent of cryptococcosis, a disease with poor patient outcomes that accounts for approximately 180,000 deaths each year. Patient outcomes may be impacted by the underlying genetics of the infecting isolate; however, our current understanding of how genetic diversity contributes to clinical outcomes is limited. Here, we leverage clinical, in vitro growth and genomic data for 284 C. neoformans isolates to identify clinically relevant pathogen variants within a population of clinical isolates from patients with human immunodeficiency virus (HIV)-associated cryptococcosis in Malawi. Through a genome-wide association study (GWAS) approach, we identify variants associated with the fungal burden and the growth rate. We also find both small and large-scale variation, including aneuploidy, associated with alternate growth phenotypes, which may impact the course of infection. Genes impacted by these variants are involved in transcriptional regulation, signal transduction, glycosylation, sugar transport, and glycolysis. We show that growth within the central nervous system (CNS) is reliant upon glycolysis in an animal model and likely impacts patient mortality, as the CNS yeast burden likely modulates patient outcome. Additionally, we find that genes with roles in sugar transport are enriched in regions under selection in specific lineages of this clinical population. Further, we demonstrate that genomic variants in two genes identified by GWAS impact virulence in animal models. Our approach identifies links between the genetic variation in C. neoformans and clinically relevant phenotypes and animal model pathogenesis, thereby shedding light on specific survival mechanisms within the CNS and identifying the pathways involved in yeast persistence. IMPORTANCE Infection outcomes for cryptococcosis, most commonly caused by C. neoformans, are influenced by host immune responses as well as by host and pathogen genetics. Infecting yeast isolates are genetically diverse; however, we lack a deep understanding of how this diversity impacts patient outcomes. To better understand both clinical isolate diversity and how diversity contributes to infection outcomes, we utilize a large collection of clinical C. neoformans samples that were isolated from patients enrolled in a clinical trial across 3 hospitals in Malawi. By combining whole-genome sequence data, clinical data, and in vitro growth data, we utilize genome-wide association approaches to examine the genetic basis of virulence. Genes with significant associations display virulence attributes in both murine and rabbit models, demonstrating that our approach can identify potential links between genetic variants and patho-biologically significant phenotypes.
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- 2022
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7. First Patient-to-Patient Intrahospital Transmission of Clade I Candida auris in France Revealed after a Two-Month Incubation Period
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Alexandre Alanio, Hannah Marie Snell, Camille Cordier, Marie Desnos-Olivier, Sarah Dellière, Nesrine Aissaoui, Aude Sturny-Leclère, Elodie Da Silva, Cyril Eblé, Martine Rouveau, Micheline Thégat, Widad Zebiche, Matthieu Lafaurie, Blandine Denis, Sophie Touratier, Mourad Benyamina, Emmanuel Dudoignon, Samia Hamane, Christina A. Cuomo, and François Dépret
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Candida auris ,burn ,ICU ,qPCR ,outbreak ,transmission ,Microbiology ,QR1-502 - Abstract
ABSTRACT Candida auris is a recently described emerging pathogen in hospital settings. Five genetic clades have been delineated, with each clade being isolated from specific geographic regions. We here describe the first transmission between 2 patients (P0 and P1) of a clade I C. auris strain imported into our burn intensive care unit from the Middle East. The strains have been investigated with whole-genome sequencing, which validated the high similarity of the genomes between isolates from P0 and P1. We repeatedly screened the two patients and contact patients (i.e., other patients present in the same hospital ward at the time of the first positive sample from P0 or P1; n = 49; 268 tests) with fungal culture and a C. auris-specific quantitative PCR assay to assess transmission patterns. We observed that P1 developed C. auris colonization between 41 and 61 days after potential exposure to P0 contamination, despite three negative screening tests as recommended by our national authorities. This study illustrates that transmission of C. auris between patients can lead to long-term incubation times before the detection of colonization. The recommended screening strategy may not be optimal and should be improved in the light of our findings. IMPORTANCE While large outbreaks of C. auris in hospital settings have been described, few clear cases of direct transmission have been documented. We here investigated the transmission of C. auris clade I between two patients with a 41- to 61-day delay between exposure and the development of colonization. This may lead to changes in the recommendations concerning treatment of C. auris cases, as an incubation period of this length is one of the first to be reported.
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- 2022
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8. Chromosome-Level Genome Assembly of a Human Fungal Pathogen Reveals Synteny among Geographically Distinct Species
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Mark Voorhies, Shirli Cohen, Terrance P. Shea, Semar Petrus, José F. Muñoz, Shane Poplawski, William E. Goldman, Todd P. Michael, Christina A. Cuomo, Anita Sil, and Sinem Beyhan
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Histoplasma capsulatum ,genome assembly ,long-read sequencing ,Microbiology ,QR1-502 - Abstract
ABSTRACT Histoplasma capsulatum, a dimorphic fungal pathogen, is the most common cause of fungal respiratory infections in immunocompetent hosts. Histoplasma is endemic in the Ohio and Mississippi River Valleys in the United States and is also distributed worldwide. Previous studies have revealed at least eight clades, each specific to a geographic location: North American classes 1 and 2 (NAm 1 and NAm 2), Latin American groups A and B (LAm A and LAm B), Eurasian, Netherlands, Australian and African, and an additional distinct lineage (H81) comprised of Panamanian isolates. Previously assembled Histoplasma genomes are highly fragmented, with the highly repetitive G217B (NAm 2) strain, which has been used for most whole-genome-scale transcriptome studies, assembled into over 250 contigs. In this study, we set out to fully assemble the repeat regions and characterize the large-scale genome architecture of Histoplasma species. We resequenced five Histoplasma strains (WU24 [NAm 1], G217B [NAm 2], H88 [African], G186AR [Panama], and G184AR [Panama]) using Oxford Nanopore Technologies long-read sequencing technology. Here, we report chromosomal-level assemblies for all five strains, which exhibit extensive synteny among the geographically distant Histoplasma isolates. The new assemblies revealed that RYP2, a major regulator of morphology and virulence, is duplicated in G186AR. In addition, we mapped previously generated transcriptome data sets onto the newly assembled chromosomes. Our analyses revealed that the expression of transposons and transposon-embedded genes are upregulated in yeast phase compared to mycelial phase in the G217B and H88 strains. This study provides an important resource for fungal researchers and further highlights the importance of chromosomal-level assemblies in analyzing high-throughput data sets. IMPORTANCE Histoplasma species are dimorphic fungi causing significant morbidity and mortality worldwide. These fungi grow as mold in the soil and as budding yeast within the human host. Histoplasma can be isolated from soil in diverse regions, including North America, South America, Africa, and Europe. Phylogenetically distinct species of Histoplasma have been isolated and sequenced. However, for the commonly used strains, genome assemblies have been fragmented, leading to underutilization of genome-scale data. This study provides chromosome-level assemblies of the commonly used Histoplasma strains using long-read sequencing technology. Comparative analysis of these genomes shows largely conserved gene order within the chromosomes. Mapping existing transcriptome data on these new assemblies reveals clustering of transcriptionally coregulated genes. The results of this study highlight the importance of obtaining chromosome-level assemblies in understanding the biology of human fungal pathogens.
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- 2022
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9. Gene Expression of Diverse Cryptococcus Isolates during Infection of the Human Central Nervous System
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Chen-Hsin Yu, Poppy Sephton-Clark, Jennifer L. Tenor, Dena L. Toffaletti, Charles Giamberardino, Miriam Haverkamp, Christina A. Cuomo, and John R. Perfect
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Cryptococcus neoformans ,meningitis ,genes ,transcription ,human disease ,Microbiology ,QR1-502 - Abstract
ABSTRACT Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.
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- 2021
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10. The Relaunch of Microbiology Spectrum
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Christina A. Cuomo
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Microbiology ,QR1-502 - Published
- 2021
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11. Genome-Wide Analysis of Experimentally Evolved Candida auris Reveals Multiple Novel Mechanisms of Multidrug Resistance
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Hans Carolus, Siebe Pierson, José F. Muñoz, Ana Subotić, Rita B. Cruz, Christina A. Cuomo, and Patrick Van Dijck
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Microbiology ,QR1-502 - Abstract
Candida aurisC. auris
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- 2021
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12. Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence
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Man Shun Fu, Livia C. Liporagi-Lopes, Samuel R. dos Santos, Jennifer L. Tenor, John R. Perfect, Christina A. Cuomo, and Arturo Casadevall
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Microbiology ,QR1-502 - Abstract
Cryptococcus neoformansC. neoformans
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- 2021
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13. Mutations in TAC1B: a Novel Genetic Determinant of Clinical Fluconazole Resistance in Candida auris
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Jeffrey M. Rybak, José F. Muñoz, Katherine S. Barker, Josie E. Parker, Brooke D. Esquivel, Elizabeth L. Berkow, Shawn R. Lockhart, Lalitha Gade, Glen E. Palmer, Theodore C. White, Steve L. Kelly, Christina A. Cuomo, and P. David Rogers
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Candida ,triazole ,resistance ,efflux ,CRISPR ,WGS ,Microbiology ,QR1-502 - Abstract
ABSTRACT Candida auris has emerged as a multidrug-resistant pathogen of great clinical concern. Approximately 90% of clinical C. auris isolates are resistant to fluconazole, the most commonly prescribed antifungal agent, and yet it remains unknown what mechanisms underpin this fluconazole resistance. To identify novel mechanisms contributing to fluconazole resistance in C. auris, fluconazole-susceptible C. auris clinical isolate AR0387 was passaged in media supplemented with fluconazole to generate derivative strains which had acquired increased fluconazole resistance in vitro. Comparative analyses of comprehensive sterol profiles, [3H]fluconazole uptake, sequencing of C. auris genes homologous to genes known to contribute to fluconazole resistance in other species of Candida, and relative expression levels of C. auris ERG11, CDR1, and MDR1 were performed. All fluconazole-evolved derivative strains were found to have acquired mutations in the zinc-cluster transcription factor-encoding gene TAC1B and to show a corresponding increase in CDR1 expression relative to the parental clinical isolate, AR0387. Mutations in TAC1B were also identified in a set of 304 globally distributed C. auris clinical isolates representing each of the four major clades. Introduction of the most common mutation found among fluconazole-resistant clinical isolates of C. auris into fluconazole-susceptible isolate AR0387 was confirmed to increase fluconazole resistance by 8-fold, and the correction of the same mutation in a fluconazole-resistant isolate, AR0390, decreased fluconazole MIC by 16-fold. Taken together, these data demonstrate that C. auris can rapidly acquire resistance to fluconazole in vitro and that mutations in TAC1B significantly contribute to clinical fluconazole resistance. IMPORTANCE Candida auris is an emerging multidrug-resistant pathogen of global concern, known to be responsible for outbreaks on six continents and to be commonly resistant to antifungals. While the vast majority of clinical C. auris isolates are highly resistant to fluconazole, an essential part of the available antifungal arsenal, very little is known about the mechanisms contributing to resistance. In this work, we show that mutations in the transcription factor TAC1B significantly contribute to clinical fluconazole resistance. These studies demonstrated that mutations in TAC1B can arise rapidly in vitro upon exposure to fluconazole and that a multitude of resistance-associated TAC1B mutations are present among the majority of fluconazole-resistant C. auris isolates from a global collection and appear specific to a subset of lineages or clades. Thus, identification of this novel genetic determinant of resistance significantly adds to the understanding of clinical antifungal resistance in C. auris.
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- 2020
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14. Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture
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Matthew C. Fisher, Sarah J. Gurr, Christina A. Cuomo, David S. Blehert, Hailing Jin, Eva H. Stukenbrock, Jason E. Stajich, Regine Kahmann, Charles Boone, David W. Denning, Neil A. R. Gow, Bruce S. Klein, James W. Kronstad, Donald C. Sheppard, John W. Taylor, Gerard D. Wright, Joseph Heitman, Arturo Casadevall, and Leah E. Cowen
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antifungal resistance ,biodiversity ,food security ,fungal pathogens ,global health ,medical mycology ,Microbiology ,QR1-502 - Abstract
ABSTRACT The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.
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- 2020
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15. Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species
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Liang Ma, Zehua Chen, Da Wei Huang, Ousmane H. Cissé, Jamie L. Rothenburger, Alice Latinne, Lisa Bishop, Robert Blair, Jason M. Brenchley, Magali Chabé, Xilong Deng, Vanessa Hirsch, Rebekah Keesler, Geetha Kutty, Yueqin Liu, Daniel Margolis, Serge Morand, Bapi Pahar, Li Peng, Koen K. A. Van Rompay, Xiaohong Song, Jun Song, Antti Sukura, Sabrina Thapar, Honghui Wang, Christiane Weissenbacher-Lang, Jie Xu, Chao-Hung Lee, Claire Jardine, Richard A. Lempicki, Melanie T. Cushion, Christina A. Cuomo, and Joseph A. Kovacs
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classification ,conserved domains ,major surface glycoprotein ,phylogenetic analysis ,Pneumocystis ,Microbiology ,QR1-502 - Abstract
ABSTRACT Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology. IMPORTANCE Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies.
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- 2020
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16. Tracing the Evolutionary History and Global Expansion of Candida auris Using Population Genomic Analyses
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Nancy A. Chow, José F. Muñoz, Lalitha Gade, Elizabeth L. Berkow, Xiao Li, Rory M. Welsh, Kaitlin Forsberg, Shawn R. Lockhart, Rodney Adam, Alexandre Alanio, Ana Alastruey-Izquierdo, Sahar Althawadi, Ana Belén Araúz, Ronen Ben-Ami, Amrita Bharat, Belinda Calvo, Marie Desnos-Ollivier, Patricia Escandón, Dianne Gardam, Revathi Gunturu, Christopher H. Heath, Oliver Kurzai, Ronny Martin, Anastasia P. Litvintseva, and Christina A. Cuomo
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Candida auris ,antifungal resistance ,emerging species ,genome analysis ,population genetics ,Microbiology ,QR1-502 - Abstract
ABSTRACT Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris. IMPORTANCE In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology.
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- 2020
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17. Tracking a Global Threat: a New Genotyping Method for Candida auris
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Christina A. Cuomo and Alexandre Alanio
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Candida auris ,genome analysis ,genotyping ,short tandem repeats ,Microbiology ,QR1-502 - Abstract
ABSTRACT Over the past decade, Candida auris has emerged as an urgent threat to public health. Initially reported from cases of ear infections in Japan and Korea, C. auris has since been detected around the world. While whole-genome sequencing has been extensively used to trace the genetic relationships of the global emergence and local outbreaks, a recent report in mBio describes a targeted genotyping method as a rapid and inexpensive method for classifying C. auris isolates (T. de Groot, Y. Puts, I. Berrio, A. Chowdhary, and J. F. Meis, mBio 11:e02971-19, https://doi.org/10.1128/mBio.02971-19, 2020).
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- 2020
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18. A New Lineage of Cryptococcus gattii (VGV) Discovered in the Central Zambezian Miombo Woodlands
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Rhys A. Farrer, Miwha Chang, Michael J. Davis, Lucy van Dorp, Dong-Hoon Yang, Terrance Shea, Thomas R. Sewell, Wieland Meyer, Francois Balloux, Hannah M. Edwards, Duncan Chanda, Geoffrey Kwenda, Mathieu Vanhove, Yun C. Chang, Christina A. Cuomo, Matthew C. Fisher, and Kyung J. Kwon-Chung
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Cryptococcus ,drug resistance evolution ,genome analysis ,molecular epidemiology ,mycology ,population genetics ,Microbiology ,QR1-502 - Abstract
ABSTRACT We discovered a new lineage of the globally important fungal pathogen Cryptococcus gattii on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of C. gattii revealed a unique suite of genes together with gene loss and inversion events. However, standard URA5 restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new URA5 RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens. IMPORTANCE Cryptococcus gattii is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that C. gattii falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety gattii five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause.
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- 2019
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19. Cryptococcus neoformans Recovered From Olive Trees (Olea europaea) in Turkey Reveal Allopatry With African and South American Lineages
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Çağri Ergin, Mustafa Şengül, Levent Aksoy, Aylin Döğen, Sheng Sun, Anna F. Averette, Christina A. Cuomo, Seyedmojtaba Seyedmousavi, Joseph Heitman, and Macit Ilkit
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Cryptococcus neoformans ,natural isolates ,Turkey ,genetic diversity ,population ,evolution ,Microbiology ,QR1-502 - Abstract
Cryptococcus species are life-threatening human fungal pathogens that cause cryptococcal meningoencephalitis in both immunocompromised and healthy hosts. The natural environmental niches of Cryptococcus include pigeon (Columba livia) guano, soil, and a variety of tree species such as Eucalyptus camaldulensis, Ceratonia siliqua, Platanus orientalis, and Pinus spp. Genetic and genomic studies of extensive sample collections have provided insights into the population distribution and composition of different Cryptococcus species in geographic regions around the world. However, few such studies examined Cryptococcus in Turkey. We sampled 388 Olea europaea (olive) and 132 E. camaldulensis trees from seven locations in coastal and inland areas of the Aegean region of Anatolian Turkey in September 2016 to investigate the distribution and genetic diversity present in the natural Cryptococcus population. We isolated 84 Cryptococcus neoformans strains (83 MATα and 1 MATa) and 3 Cryptococcus deneoformans strains (all MATα) from 87 (22.4% of surveyed) O. europaea trees; a total of 32 C. neoformans strains were isolated from 32 (24.2%) of the E. camaldulensis trees, all of which were MATα. A statistically significant difference was observed in the frequency of C. neoformans isolation between coastal and inland areas (P < 0.05). Interestingly, the MATaC. neoformans isolate was fertile in laboratory crosses with VNI and VNB MATα tester strains and produced robust hyphae, basidia, and basidiospores, thus suggesting potential sexual reproduction in the natural population. Sequencing analyses of the URA5 gene identified at least five different genotypes among the isolates. Population genetics and genomic analyses revealed that most of the isolates in Turkey belong to the VNBII lineage of C. neoformans, which is predominantly found in southern Africa; these isolates are part of a distinct minor clade within VNBII that includes several isolates from Zambia and Brazil. Our study provides insights into the geographic distribution of different C. neoformans lineages in the Mediterranean region and highlights the need for wider geographic sampling to gain a better understanding of the natural habitats, migration, epidemiology, and evolution of this important human fungal pathogen.
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- 2019
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20. Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens
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Andrew Ryan Passer, Marco A. Coelho, Robert Blake Billmyre, Minou Nowrousian, Moritz Mittelbach, Andrey M. Yurkov, Anna Floyd Averette, Christina A. Cuomo, Sheng Sun, and Joseph Heitman
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chromosomal rearrangements ,fungi ,genome analysis ,reproductive isolation ,speciation ,Microbiology ,QR1-502 - Abstract
ABSTRACT Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii. However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ∼6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus. However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established. Our study and genomic data will foster additional studies addressing fungal speciation and transitions between nonpathogenic and pathogenic Cryptococcus lineages. IMPORTANCE The evolutionary drivers of speciation are critical to our understanding of how new pathogens arise from nonpathogenic lineages and adapt to new environments. Here we focus on the Cryptococcus amylolentus species complex, a nonpathogenic fungal lineage closely related to the human-pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analyses, we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. Their genomes are ∼6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) were still able to interbreed, the resulting hybrid progeny were usually inviable or sterile, indicating that barriers to reproduction had already been established. These results advance our understanding of speciation in fungi and highlight the power of genomics in assisting our ability to correctly identify and discriminate fungal species.
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- 2019
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21. Erratum for Kim et al., 'Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance'
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Sang Hu Kim, Kali R. Iyer, Lakhansing Pardeshi, José F. Muñoz, Nicole Robbins, Christina A. Cuomo, Koon Ho Wong, and Leah E. Cowen
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Microbiology ,QR1-502 - Published
- 2019
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22. Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance
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Sang Hu Kim, Kali R. Iyer, Lakhansing Pardeshi, José F. Muñoz, Nicole Robbins, Christina A. Cuomo, Koon Ho Wong, and Leah E. Cowen
- Subjects
antifungal drug resistance ,Candida albicans ,Candida auris ,Cdr1 ,developmental program ,emerging pathogen ,Microbiology ,QR1-502 - Abstract
ABSTRACT Candida auris is an emerging fungal pathogen and a serious global health threat as the majority of clinical isolates display elevated resistance to currently available antifungal drugs. Despite the increased prevalence of C. auris infections, the mechanisms governing drug resistance remain largely elusive. In diverse fungi, the evolution of drug resistance is enabled by the essential molecular chaperone Hsp90, which stabilizes key regulators of cellular responses to drug-induced stress. Hsp90 also orchestrates temperature-dependent morphogenesis in Candida albicans, a key virulence trait. However, the role of Hsp90 in the pathobiology of C. auris remains unknown. In order to study regulatory functions of Hsp90 in C. auris, we placed HSP90 under the control of a doxycycline-repressible promoter to enable transcriptional repression. We found that Hsp90 is essential for growth in C. auris and that it enables tolerance of clinical isolates with respect to the azoles, which inhibit biosynthesis of the membrane sterol ergosterol. High-level azole resistance was independent of Hsp90 but dependent on the ABC transporter CDR1, deletion of which resulted in abrogated resistance. Strikingly, we discovered that C. auris undergoes a morphogenetic transition from yeast to filamentous growth in response to HSP90 depletion or cell cycle arrest but not in response to other cues that induce C. albicans filamentation. Finally, we observed that this developmental transition is associated with global transcriptional changes, including the induction of cell wall-related genes. Overall, this report provides a novel insight into mechanisms of drug tolerance and resistance in C. auris and describes a developmental transition in response to perturbation of a core regulator of protein homeostasis. IMPORTANCE Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90, which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90.
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- 2019
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23. Phenotypic Variability Correlates with Clinical Outcome in Cryptococcus Isolates Obtained from Botswanan HIV/AIDS Patients
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Kenya E. Fernandes, Adam Brockway, Miriam Haverkamp, Christina A. Cuomo, Floris van Ogtrop, John R. Perfect, and Dee A. Carter
- Subjects
Cryptococcus ,capsule ,cell size ,meningitis ,pleomorphism ,yeasts ,Microbiology ,QR1-502 - Abstract
ABSTRACT Pathogenic species of Cryptococcus cause hundreds of thousands of deaths annually. Considerable phenotypic variation is exhibited during infection, including increased capsule size, capsule shedding, giant cells (≥15 μm), and micro cells (≤1 μm). We examined 70 clinical isolates of Cryptococcus neoformans and Cryptococcus tetragattii from HIV/AIDS patients in Botswana to determine whether the capacity to produce morphological variants was associated with clinical parameters. Isolates were cultured under conditions designed to simulate in vivo stresses. Substantial variation was seen across morphological and clinical data. Giant cells were more common in C. tetragattii, while micro cells and shed capsule occurred in C. neoformans only. Phenotypic variables fell into two groups associated with differing symptoms. The production of “large” phenotypes (greater cell and capsule size and giant cells) was associated with higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting that these are induced in early stage infection. “Small” phenotypes (micro cells and shed capsule) were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators, and positively correlated with intracranial pressure indicators, suggesting that they are produced later during infection and may contribute to immune suppression and promote proliferation and dissemination. These trends persisted at the species level, indicating that they were not driven by association with particular Cryptococcus species. Isolates possessing giant cells, micro cells, and shed capsule were rare, but strikingly, they were associated with patient death (P = 0.0165). Our data indicate that pleomorphism is an important driver in Cryptococcus infection. IMPORTANCE Cryptococcosis results in hundreds of thousands of deaths annually, predominantly in sub-Saharan Africa. Cryptococcus is an encapsulated yeast, and during infection, cells have the capacity for substantial morphological changes, including capsule enlargement and shedding and variations in cell shape and size. In this study, we examined 70 Cryptococcus isolates causing meningitis in HIV/AIDS patients in Botswana in order to look for associations between phenotypic variation and clinical symptoms. Four variant phenotypes were seen across strains: giant cells of ≥15 µm, micro cells of ≤1 µm, shed extracellular capsule, and irregularly shaped cells. We found that “large” and “small” phenotypes were associated with differing disease symptoms, indicating that their production may be important during the disease process. Overall, our study indicates that Cryptococcus strains that can switch on cell types under different situations may be more able to sustain infection and resist the host response.
- Published
- 2018
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24. Transcriptional Heterogeneity of Cryptococcus gattii VGII Compared with Non-VGII Lineages Underpins Key Pathogenicity Pathways
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Rhys A. Farrer, Christopher B. Ford, Johanna Rhodes, Toni Delorey, Robin C. May, Matthew C. Fisher, Elaine Cloutman-Green, Francois Balloux, and Christina A. Cuomo
- Subjects
Cryptococcus ,capsule ,ergosterol ,host response ,host-pathogen interactions ,laccase ,Microbiology ,QR1-502 - Abstract
ABSTRACT Cryptococcus gattii is a pathogenic yeast of humans and other animals which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual-transcriptome sequencing (RNA-seq) experiment for four lineages of C. gattii (lineages VGI to IV) interacting with mouse macrophages at 1, 3, and 6 h postinfection. Comparisons of in vitro to ex vivo gene expression levels indicated that lineage VGII is transcriptionally divergent from non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment, and ergosterol production. Several paralogous genes demonstrated subfunctionalization between lineages, including upregulation of capsule biosynthesis-related gene CAP2 and downregulation of CAP1 in VGIII. Isolates also compensate for lineage-specific gene losses by overexpression of genetically similar paralogs, including overexpression of capsule gene CAS3 in VGIV, which have lost the CAS31 gene. Differential expression of one in five C. gattii genes was detected following coincubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and downregulation of capsule attachment genes. We also found that VGII switches expression of two laccase paralogs (from LAC1 to LAC2) during coincubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages of C. gattii by upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This report highlights the evolutionary breadth of expression profiles among the lineages of C. gattii and the diversity of transcriptional responses at this host-pathogen interface. IMPORTANCE The transcriptional profiles of related pathogens and their responses to host-induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages of C. gattii in rich media. Our analyses identified key processes, including those involving cell capsule, ergosterol production, and melanin, that are differentially expressed between lineages, and we found that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone subfunctionalization for several paralogs, including capsule biosynthesis and attachment genes. Most genes appeared downregulated during coincubation with macrophages, with the largest decrease observed for capsule attachment genes, which appeared to be coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched from expression of the laccase LAC1 to expression of LAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow-derived macrophages, marking a role in the host response to C. gattii. This work highlights the dynamic roles of key C. gattii virulence genes in response to macrophages.
- Published
- 2018
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25. Pathways of Pathogenicity: Transcriptional Stages of Germination in the Fatal Fungal Pathogen Rhizopus delemar
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Poppy C. S. Sephton-Clark, Jose F. Muñoz, Elizabeth R. Ballou, Christina A. Cuomo, and Kerstin Voelz
- Subjects
RNA-Seq ,Rhizopus delemar ,fungi ,germination ,mucormycosis ,pathogens ,Microbiology ,QR1-502 - Abstract
ABSTRACT Rhizopus delemar is an invasive fungal pathogen responsible for the frequently fatal disease mucormycosis. Germination, a crucial mechanism by which infectious spores of Rhizopus delemar cause disease, is a key developmental process that transforms the dormant spore state into a vegetative one. The molecular mechanisms that underpin this transformation may be key to controlling mucormycosis; however, the regulation of germination remains poorly understood. This study describes the phenotypic and transcriptional changes that take place over the course of germination. This process is characterized by four distinct stages: dormancy, isotropic swelling, germ tube emergence, and hyphal growth. Dormant spores are shown to be transcriptionally unique, expressing a subset of transcripts absent in later developmental stages. A large shift in the expression profile is prompted by the initiation of germination, with genes involved in respiration, chitin, cytoskeleton, and actin regulation appearing to be important for this transition. A period of transcriptional consistency can be seen throughout isotropic swelling, before the transcriptional landscape shifts again at the onset of hyphal growth. This study provides a greater understanding of the regulation of germination and highlights processes involved in transforming Rhizopus delemar from a single-cellular to multicellular organism. IMPORTANCE Germination is key to the growth of many organisms, including fungal spores. Mucormycete spores exist abundantly within the environment and germinate to form hyphae. These spores are capable of infecting immunocompromised individuals, causing the disease mucormycosis. Germination from spore to hyphae within patients leads to angioinvasion, tissue necrosis, and often fatal infections. This study advances our understanding of how spore germination occurs in the mucormycetes, identifying processes we may be able to inhibit to help prevent or treat mucormycosis.
- Published
- 2018
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26. Comparative Population Genomics Analysis of the Mammalian Fungal Pathogen Pneumocystis
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Ousmane H. Cissé, Liang Ma, Da Wei Huang, Pavel P. Khil, John P. Dekker, Geetha Kutty, Lisa Bishop, Yueqin Liu, Xilong Deng, Philippe M. Hauser, Marco Pagni, Vanessa Hirsch, Richard A. Lempicki, Jason E. Stajich, Christina A. Cuomo, and Joseph A. Kovacs
- Subjects
evolutionary biology ,genetic diversity ,genetic recombination ,pneumonia ,population structure ,Microbiology ,QR1-502 - Abstract
ABSTRACT Pneumocystis species are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable in vitro. Human Pneumocystis infections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of Pneumocystis infecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that Pneumocystis species diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of Pneumocystis populations during their spread across mammals. IMPORTANCE Understanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. Pneumocystis, a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, Pneumocystis can cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different Pneumocystis species infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that Pneumocystis may have undergone recent host shifts. The results demonstrate that Pneumocystis strains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.
- Published
- 2018
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27. Advances in Cryptococcus genomics: insights into the evolution of pathogenesis
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Christina A Cuomo, Johanna Rhodes, and Christopher A Desjardins
- Subjects
Cryptococcus neoformans ,Cryptococcus gattii ,MLST ,genome ,sequencing ,evolution ,recombination ,Microbiology ,QR1-502 ,Infectious and parasitic diseases ,RC109-216 - Abstract
Cryptococcus species are the causative agents of cryptococcal meningitis, a significant source of mortality in immunocompromised individuals. Initial work on the molecular epidemiology of this fungal pathogen utilized genotyping approaches to describe the genetic diversity and biogeography of two species, Cryptococcus neoformans and Cryptococcus gattii. Whole genome sequencing of representatives of both species resulted in reference assemblies enabling a wide array of downstream studies and genomic resources. With the increasing availability of whole genome sequencing, both species have now had hundreds of individual isolates sequenced, providing fine-scale insight into the evolution and diversification of Cryptococcus and allowing for the first genome-wide association studies to identify genetic variants associated with human virulence. Sequencing has also begun to examine the microevolution of isolates during prolonged infection and to identify variants specific to outbreak lineages, highlighting the potential role of hyper-mutation in evolving within short time scales. We can anticipate that further advances in sequencing technology and sequencing microbial genomes at scale, including metagenomics approaches, will continue to refine our view of how the evolution of Cryptococcus drives its success as a pathogen.
- Published
- 2018
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28. Microevolution of Serial Clinical Isolates of Cryptococcus neoformans var. grubii and C. gattii
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Yuan Chen, Rhys A. Farrer, Charles Giamberardino, Sharadha Sakthikumar, Alexander Jones, Timothy Yang, Jennifer L. Tenor, Omar Wagih, Marelize Van Wyk, Nelesh P. Govender, Thomas G. Mitchell, Anastasia P. Litvintseva, Christina A. Cuomo, and John R. Perfect
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.
- Published
- 2017
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29. The Case for Adopting the 'Species Complex' Nomenclature for the Etiologic Agents of Cryptococcosis
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Kyung J. Kwon-Chung, John E. Bennett, Brian L. Wickes, Wieland Meyer, Christina A. Cuomo, Kurt R. Wollenburg, Tihana A. Bicanic, Elizabeth Castañeda, Yun C. Chang, Jianghan Chen, Massimo Cogliati, Françoise Dromer, David Ellis, Scott G. Filler, Matthew C. Fisher, Thomas S. Harrison, Steven M. Holland, Shigeru Kohno, James W. Kronstad, Marcia Lazera, Stuart M. Levitz, Michail S. Lionakis, Robin C. May, Popchai Ngamskulrongroj, Peter G. Pappas, John R. Perfect, Volker Rickerts, Tania C. Sorrell, Thomas J. Walsh, Peter R. Williamson, Jianping Xu, Adrian M. Zelazny, and Arturo Casadevall
- Subjects
Cryptococcosis ,Cryptococcus gattii ,Cryptococcus neoformans ,clade ,genetic diversity ,new nomenclature ,Microbiology ,QR1-502 - Abstract
ABSTRACT Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii. Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using “Cryptococcus neoformans species complex” and “C. gattii species complex” as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.
- Published
- 2017
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30. Genome Diversity, Recombination, and Virulence across the Major Lineages of Paracoccidioides
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José F. Muñoz, Rhys A. Farrer, Christopher A. Desjardins, Juan E. Gallo, Sean Sykes, Sharadha Sakthikumar, Elizabeth Misas, Emily A. Whiston, Eduardo Bagagli, Celia M. A. Soares, Marcus de M. Teixeira, John W. Taylor, Oliver K. Clay, Juan G. McEwen, and Christina A. Cuomo
- Subjects
Paracoccidioides ,evolution ,genetic recombination ,genome analysis ,mycology ,population genetics ,Microbiology ,QR1-502 - Abstract
ABSTRACT The Paracoccidioides genus includes two species of thermally dimorphic fungi that cause paracoccidioidomycosis, a neglected health-threatening human systemic mycosis endemic to Latin America. To examine the genome evolution and the diversity of Paracoccidioides spp., we conducted whole-genome sequencing of 31 isolates representing the phylogenetic, geographic, and ecological breadth of the genus. These samples included clinical, environmental and laboratory reference strains of the S1, PS2, PS3, and PS4 lineages of P. brasiliensis and also isolates of Paracoccidioides lutzii species. We completed the first annotated genome assemblies for the PS3 and PS4 lineages and found that gene order was highly conserved across the major lineages, with only a few chromosomal rearrangements. Comparing whole-genome assemblies of the major lineages with single-nucleotide polymorphisms (SNPs) predicted from the remaining 26 isolates, we identified a deep split of the S1 lineage into two clades we named S1a and S1b. We found evidence for greater genetic exchange between the S1b lineage and all other lineages; this may reflect the broad geographic range of S1b, which is often sympatric with the remaining, largely geographically isolated lineages. In addition, we found evidence of positive selection for the GP43 and PGA1 antigen genes and genes coding for other secreted proteins and proteases and lineage-specific loss-of-function mutations in cell wall and protease genes; these together may contribute to virulence and host immune response variation among natural isolates of Paracoccidioides spp. These insights into the recent evolutionary events highlight important differences between the lineages that could impact the distribution, pathogenicity, and ecology of Paracoccidioides. IMPORTANCE Characterization of genetic differences between lineages of the dimorphic human-pathogenic fungus Paracoccidioides can identify changes linked to important phenotypes and guide the development of new diagnostics and treatments. In this article, we compared genomes of 31 diverse isolates representing the major lineages of Paracoccidioides spp. and completed the first annotated genome sequences for the PS3 and PS4 lineages. We analyzed the population structure and characterized the genetic diversity among the lineages of Paracoccidioides, including a deep split of S1 into two lineages (S1a and S1b), and differentiated S1b, associated with most clinical cases, as the more highly recombining and diverse lineage. In addition, we found patterns of positive selection in surface proteins and secreted enzymes among the lineages, suggesting diversifying mechanisms of pathogenicity and adaptation across this species complex. These genetic differences suggest associations with the geographic range, pathogenicity, and ecological niches of Paracoccidioides lineages.
- Published
- 2016
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31. Gene Expression of Diverse Cryptococcus Isolates during Infection of the Human Central Nervous System
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Jennifer L. Tenor, Chen-Hsin Yu, Poppy Sephton-Clark, Dena L. Toffaletti, Charles Giamberardino, John R. Perfect, Miriam Haverkamp, and Christina A. Cuomo
- Subjects
Central Nervous System ,Male ,Genotype ,Cryptococcus ,Virulence ,Microbiology ,Fungal Proteins ,Transcriptome ,Mice ,Meningoencephalitis ,Virology ,Gene expression ,Animals ,Humans ,RNA-Seq ,genes ,Gene ,Cryptococcus gattii ,Cryptococcus neoformans ,Genetics ,biology ,meningitis ,Cryptococcosis ,human disease ,biology.organism_classification ,QR1-502 ,Yeast ,Disease Models, Animal ,Female ,Rabbits ,transcription ,Research Article - Abstract
Cryptococcus neoformans is a major human central nervous system (CNS) fungal pathogen causing considerable morbidity and mortality. In this study, we provide the widest view to date of the yeast transcriptome directly from the human subarachnoid space and within cerebrospinal fluid (CSF). We captured yeast transcriptomes from C. neoformans of various genotypes in 31 patients with cryptococcal meningoencephalitis as well as several Cryptococcus gattii infections. Using transcriptome sequencing (RNA-seq) analyses, we compared the in vivo yeast transcriptomes to those from other environmental conditions, including in vitro growth on nutritious media or artificial CSF as well as samples collected from rabbit CSF at two time points. We ranked gene expressions and identified genetic patterns and networks across these diverse isolates that reveal an emphasis on carbon metabolism, fatty acid synthesis, transport, cell wall structure, and stress-related gene functions during growth in CSF. The most highly expressed yeast genes in human CSF included those known to be associated with survival or virulence and highlighted several genes encoding hypothetical proteins. From that group, a gene encoding the CMP1 putative glycoprotein (CNAG_06000) was selected for functional studies. This gene was found to impact the virulence of Cryptococcus in both mice and the CNS rabbit model, in agreement with a recent study also showing a role in virulence. This transcriptional analysis strategy provides a view of regulated yeast genes across genetic backgrounds important for human CNS infection and a relevant resource for the study of cryptococcal genes, pathways, and networks linked to human disease. IMPORTANCE Cryptococcus is the most common fungus causing high-morbidity and -mortality human meningitis. This encapsulated yeast has a unique propensity to travel to the central nervous system to produce disease. In this study, we captured transcriptomes of yeasts directly out of the human cerebrospinal fluid, the most concerning site of infection. By comparing the RNA transcript levels with other conditions, we gained insights into how the basic machinery involved in metabolism and environmental responses enable this fungus to cause disease at this body site. This approach was applied to clinical isolates with diverse genotypes to begin to establish a genotype-agnostic understanding of how the yeast responds to stress. Based on these results, future studies can focus on how these genes and their pathways and networks can be targeted with new therapeutics and possibly classify yeasts with bad infection outcomes.
- Published
- 2021
32. Genome Evolution and Innovation across the Four Major Lineages of Cryptococcus gattii
- Author
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Rhys A. Farrer, Christopher A. Desjardins, Sharadha Sakthikumar, Sharvari Gujja, Sakina Saif, Qiandong Zeng, Yuan Chen, Kerstin Voelz, Joseph Heitman, Robin C. May, Matthew C. Fisher, and Christina A. Cuomo
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT Cryptococcus gattii is a fungal pathogen of humans, causing pulmonary infections in otherwise healthy hosts. To characterize genomic variation among the four major lineages of C. gattii (VGI, -II, -III, and -IV), we generated, annotated, and compared 16 de novo genome assemblies, including the first for the rarely isolated lineages VGIII and VGIV. By identifying syntenic regions across assemblies, we found 15 structural rearrangements, which were almost exclusive to the VGI-III-IV lineages. Using synteny to inform orthology prediction, we identified a core set of 87% of C. gattii genes present as single copies in all four lineages. Remarkably, 737 genes are variably inherited across lineages and are overrepresented for response to oxidative stress, mitochondrial import, and metal binding and transport. Specifically, VGI has an expanded set of iron-binding genes thought to be important to the virulence of Cryptococcus, while VGII has expansions in the stress-related heat shock proteins relative to the other lineages. We also characterized genes uniquely absent in each lineage, including a copper transporter absent from VGIV, which influences Cryptococcus survival during pulmonary infection and the onset of meningoencephalitis. Through inclusion of population-level data for an additional 37 isolates, we identified a new transcontinental clonal group that we name VGIIx, mitochondrial recombination between VGII and VGIII, and positive selection of multidrug transporters and the iron-sulfur protein aconitase along multiple branches of the phylogenetic tree. Our results suggest that gene expansion or contraction and positive selection have introduced substantial variation with links to mechanisms of pathogenicity across this species complex. IMPORTANCE The genetic differences between phenotypically different pathogens provide clues to the underlying mechanisms of those traits and can lead to new drug targets and improved treatments for those diseases. In this paper, we compare 16 genomes belonging to four highly differentiated lineages of Cryptococcus gattii, which cause pulmonary infections in otherwise healthy humans and other animals. Half of these lineages have not had their genomes previously assembled and annotated. We identified 15 ancestral rearrangements in the genome and over 700 genes that are unique to one or more lineages, many of which are associated with virulence. In addition, we found evidence for recent transcontinental spread, mitochondrial genetic exchange, and positive selection in multidrug transporters. Our results suggest that gene expansion/contraction and positive selection are diversifying the mechanisms of pathogenicity across this species complex.
- Published
- 2015
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33. Analysis of a Food-Borne Fungal Pathogen Outbreak: Virulence and Genome of a Mucor circinelloides Isolate from Yogurt
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Soo Chan Lee, R. Blake Billmyre, Alicia Li, Sandra Carson, Sean M. Sykes, Eun Young Huh, Piotr Mieczkowski, Dennis C. Ko, Christina A. Cuomo, and Joseph Heitman
- Subjects
Microbiology ,QR1-502 - Abstract
ABSTRACT Food-borne pathogens are ongoing problems, and new pathogens are emerging. The impact of fungi, however, is largely underestimated. Recently, commercial yogurts contaminated with Mucor circinelloides were sold, and >200 consumers became ill with nausea, vomiting, and diarrhea. Mucoralean fungi cause the fatal fungal infection mucormycosis, whose incidence has been continuously increasing. In this study, we isolated an M. circinelloides strain from a yogurt container, and multilocus sequence typing identified the strain as Mucor circinelloides f. circinelloides. M. circinelloides f. circinelloides is the most virulent M. circinelloides subspecies and is commonly associated with human infections, whereas M. circinelloides f. lusitanicus and M. circinelloides f. griseocyanus are less common causes of infection. Whole-genome analysis of the yogurt isolate confirmed it as being close to the M. circinelloides f. circinelloides subgroup, with a higher percentage of divergence with the M. circinelloides f. lusitanicus subgroup. In mating assays, the yogurt isolate formed sexual zygospores with the (−) M. circinelloides f. circinelloides tester strain, which is congruent with its sex locus encoding SexP, the (+) mating type sex determinant. The yogurt isolate was virulent in murine and wax moth larva host systems. In a murine gastromucormycosis model, Mucor was recovered from fecal samples of infected mice for up to 10 days, indicating that Mucor can survive transit through the GI tract. In interactions with human immune cells, M. circinelloides f. lusitanicus induced proinflammatory cytokines but M. circinelloides f. circinelloides did not, which may explain the different levels of virulence in mammalian hosts. This study demonstrates that M. circinelloides can spoil food products and cause gastrointestinal illness in consumers and may pose a particular risk to immunocompromised patients. IMPORTANCE The U.S. FDA reported that yogurt products were contaminated with M. circinelloides, a mucoralean fungal pathogen, and >200 consumers complained of symptoms, including vomiting, nausea, and diarrhea. The manufacturer voluntarily withdrew the affected yogurt products from the market. Compared to other food-borne pathogens, including bacteria, viruses, and parasites, less focus has been placed on the risk of fungal pathogens. This study evaluates the potential risk from the food-borne fungal pathogen M. circinelloides that was isolated from the contaminated commercial yogurt. We successfully cultured an M. circinelloides isolate and found that the isolate belongs to the species M. circinelloides f. circinelloides, which is often associated with human infections. In murine and insect host models, the isolate was virulent. While information disseminated in the popular press would suggest this fungal contaminant poses little or no risk to consumers, our results show instead that it is capable of causing significant infections in animals.
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- 2014
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34. Highly Recombinant VGII Cryptococcus gattii Population Develops Clonal Outbreak Clusters through both Sexual Macroevolution and Asexual Microevolution
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R. Blake Billmyre, Daniel Croll, Wenjun Li, Piotr Mieczkowski, Dee A. Carter, Christina A. Cuomo, James W. Kronstad, and Joseph Heitman
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Microbiology ,QR1-502 - Abstract
ABSTRACT An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV+ patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors. IMPORTANCE Cryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in the 1990s and remains elevated. These infections are attributable to three clonal lineages of C. gattii, VGIIa, VGIIb, and VGIIc, with only VGIIa identified once previously in the Pacific Northwest prior to the start of the outbreak, albeit in a less virulent form. This study addresses the origin and emergence of this outbreak, using whole-genome sequencing and comparison of both outbreak and global isolates. We show that VGIIa arose mitotically from a less virulent clonal group, possibly via the action of a mutator phenotype, while VGIIb was likely introduced from Australia, and VGIIc appears to have emerged in the United States or in an undersampled locale via sexual reproduction. This work shows that multiple processes can contribute to the emergence of an outbreak.
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- 2014
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35. Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence
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Jennifer L. Tenor, Arturo Casadevall, Samuel R. dos Santos Júnior, John R. Perfect, Christina A. Cuomo, Livia C. Liporagi-Lopes, and Man Shun Fu
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Virulence ,Human pathogen ,Moths ,Microbiology ,Amoeba (operating system) ,opportunistic fungi ,Phagocytosis ,Virology ,evolution ,host-pathogen interactions ,Animals ,Humans ,amoeba ,Gene ,Cryptococcus neoformans ,Acanthamoeba castellanii ,Phagocytes ,biology ,Macrophages ,Cryptococcosis ,biology.organism_classification ,Phenotype ,QR1-502 ,Mice, Inbred C57BL ,Larva ,Cytokines ,Protozoa ,Female ,Research Article - Abstract
Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle., Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG_03013; OPT1) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG_00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation.
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- 2021
36. Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection
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Diego A. Martinez, Brian G. Oliver, Yvonne Gräser, Jonathan M. Goldberg, Wenjun Li, Nilce M. Martinez-Rossi, Michel Monod, Ekaterina Shelest, Richard C. Barton, Elizabeth Birch, Axel A. Brakhage, Zehua Chen, Sarah J. Gurr, David Heiman, Joseph Heitman, Idit Kosti, Antonio Rossi, Sakina Saif, Marketa Samalova, Charles W. Saunders, Terrance Shea, Richard C. Summerbell, Jun Xu, Sarah Young, Qiandong Zeng, Bruce W. Birren, Christina A. Cuomo, and Theodore C. White
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Microbiology ,QR1-502 - Abstract
ABSTRACT The major cause of athlete’s foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response. IMPORTANCE Athlete’s foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete’s foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host’s immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.
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- 2012
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37. Threats Posed by the Fungal Kingdom to Humans, Wildlife, and Agriculture
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Sarah J. Gurr, Gerard D. Wright, Donald C. Sheppard, Christina A. Cuomo, Leah E. Cowen, David S. Blehert, Hailing Jin, James W. Kronstad, Arturo Casadevall, Eva H. Stukenbrock, Charles Boone, Matthew C. Fisher, Regine Kahmann, Joseph Heitman, Neil A. R. Gow, Jason E. Stajich, David W. Denning, John W. Taylor, Bruce S. Klein, and Chowdhary, Anuradha
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Ecology (disciplines) ,Wildlife ,Biodiversity ,Wild ,global health ,Animals, Wild ,Biology ,Microbiology ,Host-Microbe Biology ,03 medical and health sciences ,Virology ,Global health ,Animals ,Humans ,Microbiome ,Plant Diseases ,biodiversity ,030304 developmental biology ,0303 health sciences ,Food security ,030306 microbiology ,Agroforestry ,business.industry ,Fungi ,plant-pathogenic fungi ,Agriculture ,food security ,Plants ,antifungal resistance ,QR1-502 ,Plant disease ,Infectious Diseases ,Emerging Infectious Diseases ,Good Health and Well Being ,Mycoses ,wildlife pathogens ,Minireview ,fungal pathogens ,Infection ,business ,medical mycology - Abstract
The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals., The fungal kingdom includes at least 6 million eukaryotic species and is remarkable with respect to its profound impact on global health, biodiversity, ecology, agriculture, manufacturing, and biomedical research. Approximately 625 fungal species have been reported to infect vertebrates, 200 of which can be human associated, either as commensals and members of our microbiome or as pathogens that cause infectious diseases. These organisms pose a growing threat to human health with the global increase in the incidence of invasive fungal infections, prevalence of fungal allergy, and the evolution of fungal pathogens resistant to some or all current classes of antifungals. More broadly, there has been an unprecedented and worldwide emergence of fungal pathogens affecting animal and plant biodiversity. Approximately 8,000 species of fungi and Oomycetes are associated with plant disease. Indeed, across agriculture, such fungal diseases of plants include new devastating epidemics of trees and jeopardize food security worldwide by causing epidemics in staple and commodity crops that feed billions. Further, ingestion of mycotoxins contributes to ill health and causes cancer. Coordinated international research efforts, enhanced technology translation, and greater policy outreach by scientists are needed to more fully understand the biology and drivers that underlie the emergence of fungal diseases and to mitigate against their impacts. Here, we focus on poignant examples of emerging fungal threats in each of three areas: human health, wildlife biodiversity, and food security.
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- 2020
38. Tracing the Evolutionary History and Global Expansion of Candida auris Using Population Genomic Analyses
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Ana Belén Araúz, Elizabeth L. Berkow, Oliver Kurzai, Christopher H. Heath, Rodney Adam, Sahar Althawadi, Xiao Li, Shawn R. Lockhart, Patricia Escandón, Ronen Ben-Ami, Anastasia P. Litvintseva, Lalitha Gade, Alexandre Alanio, Revathi Gunturu, Kaitlin Forsberg, Ana Alastruey-Izquierdo, Nancy A. Chow, Christina A. Cuomo, Amrita Bharat, Marie Desnos-Ollivier, Dianne Gardam, Belinda Calvo, Ronny Martin, José F. Muñoz, Rory M. Welsh, Centers for Disease Control and Prevention (CDC), Broad Institute [Cambridge], Massachusetts Institute of Technology (MIT)-Harvard University [Cambridge], Aga Khan University Hospital (AKUH), Nairobi, Mycologie moléculaire - Molecular Mycology, Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Centre National de Référence Mycoses Invasives et Antifongiques - National Reference Center Invasive Mycoses & Antifungals (CNRMA), Institut Pasteur [Paris], Laboratoire de Parasitologie-Mycologie [CHU Saint Louis, Paris], Groupe Hospitalier Saint Louis - Lariboisière - Fernand Widal [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Université de Paris (UP), Instituto de Salud Carlos III [Madrid] (ISC), King Faisal Specialist Hospital and Research Centre, Riyadh, Hospital Santo Tomás, Tel Aviv Sourasky Medical Center [Te Aviv], Tel Aviv University [Tel Aviv], Public Health Agency of Canada (PHAC), Universidad del Zulia (LUZ), Instituto Nacional de Salud [Bogota], Fiona Stanley Hospital [Murdoch], Royal Perth Hospital, The University of Western Australia (UWA), Leibniz Institute for Natural Product Research and Infection Biology (Hans Knoell Institute), University of Würzburg = Universität Würzburg, This project has been funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under award U19AI110818 to the Broad Institute. C.A.C. is a CIFAR fellow in the Fungal Kingdom Program. This work was also made possible through support from the Advanced Molecular Detection (AMD) initiative at CDC., National Institute of Allergy and Infectious Diseases (United States), Harvard University-Massachusetts Institute of Technology (MIT), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP), Université Paris Cité (UPCité), King Faisal Specialist Hospital and Research Centre (KFSH & RC), and Tel Aviv University (TAU)
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Azoles ,Antifungal Agents ,Population genetics ,Emerging species ,Echinocandins ,MESH: Azoles ,Molecular clock ,Clade ,MESH: Phylogeny ,Fluconazole ,Phylogeny ,[SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology ,Candida ,genome analysis ,emerging species ,0303 health sciences ,education.field_of_study ,Molecular Epidemiology ,Candida auris ,antifungal resistance ,Biological Evolution ,QR1-502 ,3. Good health ,Phylogeography ,MESH: Phylogeography ,MESH: Genome, Fungal ,MESH: Fluconazole ,Genome, Fungal ,MESH: Metagenomics ,MESH: Whole Genome Sequencing ,Research Article ,MESH: Mutation ,Population ,Genes, Fungal ,MESH: Genetics, Population ,Context (language use) ,MESH: Biological Evolution ,Ecological and Evolutionary Science ,Biology ,Microbiology ,MESH: Drug Resistance, Fungal ,03 medical and health sciences ,Phylogenetics ,Drug Resistance, Fungal ,MESH: Candida ,Virology ,Humans ,MESH: Molecular Epidemiology ,Candidiasis, Invasive ,education ,030304 developmental biology ,MESH: Humans ,Molecular epidemiology ,Whole Genome Sequencing ,030306 microbiology ,MESH: Echinocandins ,population genetics ,Antifungal resistance ,Genome analysis ,MESH: Antifungal Agents ,MESH: Candidiasis, Invasive ,Genetics, Population ,Evolutionary biology ,Mutation ,Metagenomics ,MESH: Genes, Fungal - Abstract
In less than a decade, C. auris has emerged in health care settings worldwide; this species is capable of colonizing skin and causing outbreaks of invasive candidiasis. In contrast to other Candida species, C. auris is unique in its ability to spread via nosocomial transmission and its high rates of drug resistance. As part of the public health response, whole-genome sequencing has played a major role in characterizing transmission dynamics and detecting new C. auris introductions. Through a global collaboration, we assessed genome evolution of isolates of C. auris from 19 countries. Here, we described estimated timing of the expansion of each C. auris clade and of fluconazole resistance, characterized discrete phylogeographic population structure of each clade, and compared genome data to sensitivity measurements to describe how antifungal resistance mechanisms vary across the population. These efforts are critical for a sustained, robust public health response that effectively utilizes molecular epidemiology., Candida auris has emerged globally as a multidrug-resistant yeast that can spread via nosocomial transmission. An initial phylogenetic study of isolates from Japan, India, Pakistan, South Africa, and Venezuela revealed four populations (clades I, II, III, and IV) corresponding to these geographic regions. Since this description, C. auris has been reported in more than 30 additional countries. To trace this global emergence, we compared the genomes of 304 C. auris isolates from 19 countries on six continents. We found that four predominant clades persist across wide geographic locations. We observed phylogeographic mixing in most clades; clade IV, with isolates mainly from South America, demonstrated the strongest phylogeographic substructure. C. auris isolates from two clades with opposite mating types were detected contemporaneously in a single health care facility in Kenya. We estimated a Bayesian molecular clock phylogeny and dated the origin of each clade within the last 360 years; outbreak-causing clusters from clades I, III, and IV originated 36 to 38 years ago. We observed high rates of antifungal resistance in clade I, including four isolates resistant to all three major classes of antifungals. Mutations that contribute to resistance varied between the clades, with Y132F in ERG11 as the most widespread mutation associated with azole resistance and S639P in FKS1 for echinocandin resistance. Copy number variants in ERG11 predominantly appeared in clade III and were associated with fluconazole resistance. These results provide a global context for the phylogeography, population structure, and mechanisms associated with antifungal resistance in C. auris.
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- 2020
39. Diversity and Complexity of the Large Surface Protein Family in the Compacted Genomes of Multiple Pneumocystis Species
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Koen K. A. Van Rompay, Yueqin Liu, Honghui Wang, Daniel Margolis, Jun Song, Robert V Blair, Richard A. Lempicki, Chao-Hung Lee, Christiane Weissenbacher-Lang, Bapi Pahar, Claire M. Jardine, Xiaohong Song, Geetha Kutty, Liang Ma, Vanessa M. Hirsch, Joseph A. Kovacs, Xilong Deng, Alice Latinne, Jamie L. Rothenburger, Antti Sukura, Jason M. Brenchley, Sabrina Thapar, Rebekah I. Keesler, Serge Morand, Li Peng, Melanie T. Cushion, Da-Wei Huang, Zehua Chen, Jie Xu, Lisa R. Bishop, Christina A. Cuomo, Ousmane H. Cissé, Magali Chabé, Institut des Sciences de l'Evolution de Montpellier (UMR ISEM), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-École pratique des hautes études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université de Montpellier (UM)-Institut de recherche pour le développement [IRD] : UR226-Centre National de la Recherche Scientifique (CNRS), Veterinary Biosciences, Helsinki One Health (HOH), Antti Sukura / Principal Investigator, Veterinary Pathology and Parasitology, and Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-École Pratique des Hautes Études (EPHE)
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[SDV]Life Sciences [q-bio] ,Disease ,Pneumocystis pneumonia ,Genome ,ANTIGENIC VARIATION ,Phylogeny ,Immunodeficiency ,Mammals ,Genetics ,11832 Microbiology and virology ,0303 health sciences ,Membrane Glycoproteins ,1184 Genetics, developmental biology, physiology ,GLYCOPROTEIN ,RABBITS ,QR1-502 ,3. Good health ,classification ,EXPRESSION SITE ,major surface glycoprotein ,Genome, Fungal ,Research Article ,GENES ,Biology ,Microbiology ,SEQUENCE ,Host-Microbe Biology ,Evolution, Molecular ,Fungal Proteins ,03 medical and health sciences ,Phylogenetics ,Sequence Homology, Nucleic Acid ,Virology ,Antigenic variation ,medicine ,Animals ,WILD NORWAY RATS ,Gene ,030304 developmental biology ,IDENTIFICATION ,030306 microbiology ,Pneumocystis ,phylogenetic analysis ,Genetic Variation ,medicine.disease ,Rats ,SP NOV ,CARINII ,Adaptation ,conserved domains - Abstract
Pneumocystis continues to be a major cause of disease in humans with immunodeficiency, especially those with HIV/AIDS and organ transplants, and is being seen with increasing frequency worldwide in patients treated with immunodepleting monoclonal antibodies. Annual health care associated with Pneumocystis pneumonia costs ∼$475 million dollars in the United States alone. In addition to causing overt disease in immunodeficient individuals, Pneumocystis can cause subclinical infection or colonization in healthy individuals, which may play an important role in species preservation and disease transmission. Our work sheds new light on the diversity and complexity of the msg superfamily and strongly suggests that the versatility of this superfamily reflects multiple functions, including antigenic variation to allow immune evasion and optimal adaptation to host environmental conditions to promote efficient infection and transmission. These findings are essential to consider in developing new diagnostic and therapeutic strategies., Pneumocystis, a major opportunistic pathogen in patients with a broad range of immunodeficiencies, contains abundant surface proteins encoded by a multicopy gene family, termed the major surface glycoprotein (Msg) gene superfamily. This superfamily has been identified in all Pneumocystis species characterized to date, highlighting its important role in Pneumocystis biology. In this report, through a comprehensive and in-depth characterization of 459 msg genes from 7 Pneumocystis species, we demonstrate, for the first time, the phylogeny and evolution of conserved domains in Msg proteins and provide a detailed description of the classification, unique characteristics, and phylogenetic relatedness of five Msg families. We further describe, for the first time, the relative expression levels of individual msg families in two rodent Pneumocystis species, the substantial variability of the msg repertoires in P. carinii from laboratory and wild rats, and the distinct features of the expression site for the classic msg genes in Pneumocystis from 8 mammalian host species. Our analysis suggests multiple functions for this superfamily rather than just conferring antigenic variation to allow immune evasion as previously believed. This study provides a rich source of information that lays the foundation for the continued experimental exploration of the functions of the Msg superfamily in Pneumocystis biology.
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- 2020
40. A New Lineage of <named-content content-type='genus-species'>Cryptococcus gattii</named-content> (VGV) Discovered in the Central Zambezian Miombo Woodlands
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Kyung J. Kwon-Chung, Wieland Meyer, Lucy van Dorp, Miwha Chang, Yun C. Chang, Rhys A. Farrer, Hannah M. Edwards, Terrance Shea, Francois Balloux, Matthew C. Fisher, Christina A. Cuomo, Dong-Hoon Yang, Mathieu Vanhove, Thomas R. Sewell, Duncan Chanda, Michael Davis, Geoffrey Kwenda, and Medical Research Council (MRC)
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Lineage (evolution) ,Population genetics ,SEROTYPE-A ,Forests ,SUSCEPTIBILITY ,ANNOTATION ,molecular epidemiology ,Animal Diseases ,Environmental Microbiology ,Phylogeny ,Soil Microbiology ,POPULATION ,genome analysis ,0303 health sciences ,education.field_of_study ,biology ,Phylogenetic tree ,Genomics ,QR1-502 ,Phenotype ,DECAYED WOOD ,Filobasidiella ,Restriction fragment length polymorphism ,Genome, Fungal ,Life Sciences & Biomedicine ,Research Article ,0605 Microbiology ,DATABASE ,Population ,Zambia ,Ecological and Evolutionary Science ,Microbiology ,FILOBASIDIELLA ,03 medical and health sciences ,Virology ,parasitic diseases ,NEOFORMANS VAR. GRUBII ,Animals ,education ,Cryptococcus gattii ,030304 developmental biology ,Plant Diseases ,Science & Technology ,Molecular epidemiology ,030306 microbiology ,population genetics ,biology.organism_classification ,bacterial infections and mycoses ,EVOLUTION ,Cryptococcus ,Evolutionary biology ,drug resistance evolution ,mycology ,VIRULENCE - Abstract
Cryptococcus gattii is an environmental pathogen that causes severe systemic infection in immunocompetent individuals more often than in immunocompromised humans. Over the past 2 decades, researchers have shown that C. gattii falls within four genetically distinct major lineages. By combining field work from an understudied ecological region (the Central Miombo Woodlands of Zambia, Africa), genome sequencing and assemblies, phylogenetic and population genetic analyses, and phenotypic characterization (morphology, histopathological, drug-sensitivity, survival experiments), we discovered a hitherto unknown lineage, which we name VGV (variety gattii five). The discovery of a new lineage from an understudied ecological region has far-reaching implications for the study and understanding of fungal pathogens and diseases they cause., We discovered a new lineage of the globally important fungal pathogen Cryptococcus gattii on the basis of analysis of six isolates collected from three locations spanning the Central Miombo Woodlands of Zambia, Africa. All isolates were from environments (middens and tree holes) that are associated with a small mammal, the African hyrax. Phylogenetic and population genetic analyses confirmed that these isolates form a distinct, deeply divergent lineage, which we name VGV. VGV comprises two subclades (A and B) that are capable of causing mild lung infection with negligible neurotropism in mice. Comparing the VGV genome to previously identified lineages of C. gattii revealed a unique suite of genes together with gene loss and inversion events. However, standard URA5 restriction fragment length polymorphism (RFLP) analysis could not distinguish between VGV and VGIV isolates. We therefore developed a new URA5 RFLP method that can reliably identify the newly described lineage. Our work highlights how sampling understudied ecological regions alongside genomic and functional characterization can broaden our understanding of the evolution and ecology of major global pathogens.
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- 2019
41. Genetic and Genomic Analyses Reveal Boundaries between Species Closely Related to Cryptococcus Pathogens
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Christina A. Cuomo, Marco A. Coelho, Anna F. Averette, Sheng Sun, Andrew Ryan Passer, Minou Nowrousian, Moritz Mittelbach, Joseph Heitman, R.B. Billmyre, and Andrey Yurkov
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Species complex ,reproductive isolation ,Lineage (evolution) ,Species distribution ,Cryptococcus ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Virology ,Ectopic recombination ,Cryptococcus gattii ,chromosomal rearrangements ,genome analysis ,030304 developmental biology ,Cryptococcus neoformans ,0303 health sciences ,Phylogenetic tree ,biology ,030306 microbiology ,Reproductive isolation ,biology.organism_classification ,QR1-502 ,Sexual reproduction ,speciation ,Evolutionary biology ,fungi ,030217 neurology & neurosurgery - Abstract
Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes over 200,000 new infections in humans annually. The closest non-human pathogenic relatives are the sibling species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii. However, because relatively few isolates of each species are available, it is unclear whether they represent divergent lineages of the same species or different biological species. The recent isolation of an additional strain, preliminarily identified as T. wingfieldii, prompted us to reexamine this group as it may inform about the evolutionary processes underlying the diversification of both non-pathogenic and pathogenic Cryptococcus lineages. Using genomic data, we reappraised the phylogenetic relationship of the four available strains and confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. Comparison of full-length chromosome assemblies revealed approximately 6% pairwise sequence divergence between the three species, and identified significant genomic changes, including inversions as well as a reciprocal translocation that involved inter-centromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can undergo sexual reproduction with C. amylolentus. However, most of the spores resulting from this cross were inviable, and many were sterile, indicating that the two species are genetically isolated through intrinsic post-zygotic barriers and possibly due to niche differentiation. Genome sequencing and analysis of the progeny demonstrated decreased recombination frequency during meiosis in heterospecific crosses compared to C. amylolentus conspecific crosses. This study advances our understanding of speciation in fungi and highlights the power of genomics in assisting our ability to correctly identify and discriminate fungal species.Author SummaryThe idea of species as discrete natural units seems rather intuitive for most people, just as cells are the basic units of life. However, when observing variation across a species range, boundaries can become blurred making it less than obvious when different populations evolve into separate species. Additionally, separate species can still interbreed, such as lions breeding with tigers to produce a liger or a tigon (depending on the paternal and maternal species of origin), but the resulting offspring is usually inviable or sterile, which in turn is evidence that the parents involved are distinct species. Therefore, what species are and how they originate is still an open question in evolutionary biology. While recent advances have been made in the fields of animal and plant speciation, many other important components of biological diversity, such as fungi, are still understudied. Genome sequencing is now providing new tools to address the genetic mechanisms that drive divergence and reproductive isolation between populations, including genetic incompatibilities, sequence divergence, and chromosomal rearrangements. Here we focus on the Cryptococcus amylolentus species complex, a non-pathogenic fungal lineage closely related to the human pathogenic Cryptococcus neoformans/Cryptococcus gattii complex. Using genetic and genomic analysis we reexamined the species boundaries of four available isolates within the C. amylolentus complex and revealed three genetically isolated species. The genomes of these species are ~6% divergent and exhibit chromosome rearrangements, including translocations and small-scale inversions. Although two of the species (C. amylolentus and newly described C. floricola) are still able to interbreed, the resulting hybrid progeny were mostly inviable, and many were sterile, indicating that barriers to reproduction have already been established. Our results will foster additional studies addressing the transitions between non-pathogenic and pathogenic Cryptococcus lineages.
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- 2019
42. Erratum for Kim et al., 'Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance'
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Christina A. Cuomo, Sang Hu Kim, José F. Muñoz, Lakhansing Pardeshi, Kali R. Iyer, Koon Ho Wong, Leah E. Cowen, and Nicole Robbins
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chemistry.chemical_classification ,0303 health sciences ,biology ,030306 microbiology ,Morphogenesis ,Azole resistance ,biology.organism_classification ,Microbiology ,Molecular biology ,Hsp90 ,Genetic analysis ,QR1-502 ,Corpus albicans ,03 medical and health sciences ,chemistry ,Candida auris ,Virology ,biology.protein ,Azole ,Candida albicans ,030304 developmental biology - Abstract
Volume 10, no. 1, e02529-18, 2019, [https://doi.org/10.1128/mBio.02529-18][1]. [Figure 1A][2] spotting images were incorrectly duplicated for the Candida albicans and Candida auris plates. We confirmed the essentiality of HSP90 expression in C. albicans and C. auris with multiple biological
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- 2019
43. Transcriptional heterogeneity of Cryptococcus gattii VGII compared with non-VGII lineages underpins key pathogenicity pathways
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Elaine Cloutman-Green, Christina A. Cuomo, Matthew C. Fisher, Francois Balloux, Toni Delorey, Johanna Rhodes, Rhys A. Farrer, Christopher B. Ford, and Robin C. May
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0301 basic medicine ,Bacterial capsule ,Lineage (evolution) ,capsule ,030106 microbiology ,Virulence ,Microbiology ,laccase ,03 medical and health sciences ,Immune system ,Downregulation and upregulation ,Gene expression ,host response ,host-pathogen interactions ,Molecular Biology ,Cryptococcus gattii ,Gene ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,ergosterol ,030306 microbiology ,biology.organism_classification ,Yeast ,QR1-502 ,Cryptococcus ,030104 developmental biology ,Subfunctionalization ,FOSB - Abstract
Cryptococcus gattiiis a pathogenic yeast of humans and other animals, which causes disease predominantly in immunocompetent hosts. Infection begins when aerosolized yeast or spores enter the body, triggering an immune response, including engulfment by macrophages. To understand the early transcriptional signals in both the yeast and its mammalian host, we performed a time-course dual RNA-seq experiment for four lineages ofC. gattii(VGI-IV) interacting with mouse macrophages at 1hr, 3hr and 6hr post infection. Comparison ofin vitrotoex vivogene expression indicates lineage VGII is transcriptionally divergent to non-VGII lineages, including differential expression of genes involved in capsule synthesis, capsule attachment and ergosterol production. Various paralogs demonstrate sub-functionalisation between lineages including an upregulation of capsule biosynthesis-related geneCAP2, and downregulation ofCAP1in VGIII. Isolates also compensate for lineage-specific gene-losses by over-expression of genetically similar paralogs, including an over-expression of capsule geneCAS3in VGIV having lostCAS31. Differential expression of one in fiveC. gattiigenes was detected following co-incubation with mouse macrophages; all isolates showed high induction of oxidative-reduction functions and a downregulation of capsule attachment genes. We also show that VGII switches expression of two laccase paralogs (fromLAC1toLAC2) during co-incubation of macrophages. Finally, we found that mouse macrophages respond to all four lineages ofC. gattiiby upregulating FosB/Jun/Egr1 regulatory proteins at early time points. This study highlights the evolutionary breadth of expression profiles amongst the lineages ofC. gattiiand the diversity of transcriptional responses at this host-pathogen interface.ImportanceThe transcriptional profiles of related pathogens and their response to host induced stresses underpin their pathogenicity. Expression differences between related pathogens during host interaction can indicate when and how these genes contribute to virulence, ultimately informing new and improved treatment strategies for those diseases. In this paper, we compare the transcriptional profiles of five isolates representing four lineages ofC. gattiiin rich media. Our analyses identified key processes including cell capsule, ergosterol production and melanin that are differentially expressed between lineages, and we find that VGII has the most distinct profile in terms of numbers of differentially expressed genes. All lineages have also undergone sub-functionalisation for various paralogs including capsule biosynthesis and attachment genes. Most genes appeared down-regulated during co-incubation with macrophages, with the largest decrease observed for capsule attachment genes, which appears coordinated with a stress response, as all lineages also upregulated oxidative stress response genes. Furthermore, VGII upregulated many genes that are linked to ergosterol biosynthesis and switched expression of the laccaseLAC1toLAC2 ex vivo. Finally, we saw a pronounced increase in the FosB/Jun/Egr1 regulatory proteins at early time points in bone marrow derived macrophages, marking a role in the host response toC. gattii. This work highlights the dynamic roles of keyC. gattiivirulence genes in response to macrophages.
- Published
- 2018
44. Phenotypic variability correlates with clinical outcome inCryptococcusisolates obtained from Botswanan HIV/AIDS patients
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Dee A. Carter, Kenya E. Fernandes, Christina A. Cuomo, Adam Brockway, Floris van Ogtrop, Miriam Haverkamp, and John R. Perfect
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0301 basic medicine ,Cell type ,capsule ,Cell ,Cryptococcus ,yeasts ,Microbiology ,03 medical and health sciences ,Immune system ,Virology ,medicine ,030304 developmental biology ,Cryptococcus neoformans ,0303 health sciences ,biology ,030306 microbiology ,meningitis ,Capsule ,pleomorphism ,medicine.disease ,biology.organism_classification ,Phenotype ,QR1-502 ,3. Good health ,cell size ,030104 developmental biology ,medicine.anatomical_structure ,Pleomorphism (cytology) ,Giant cell ,Cryptococcosis ,Meningitis - Abstract
Pathogenic species ofCryptococcuscause hundreds of thousands of deaths annually. Considerable phenotypic variation is exhibited during infection, including increased capsule size, capsule shedding, giant cells (≥ 15 μm) and micro cells (≤ 1 μm). We examined 70 clinical isolates ofCryptococcus neoformansandCryptococcus tetragattiifrom HIV/AIDS patients in Botswana to determine if the capacity to produce morphological variants was associated with clinical parameters. Isolates were cultured under conditions designed to simulatein vivostresses. Substantial variation was seen across morphological and clinical data. Giant cells were more common inC. tetragattii,while micro cells and shed capsule occurred inC. neoformansonly. Phenotypic variables fell into two groups associated with differing symptoms. The production of “large” phenotypes (greater cell and capsule size and giant cells) was associated with higher CD4 count and was negatively correlated with intracranial pressure indicators, suggesting these are induced in early-stage infection. “Small” phenotypes (micro cells and shed capsule) were associated with lower CD4 counts, negatively correlated with meningeal inflammation indicators and positively correlated with intracranial pressure indicators, suggesting they are produced later during infection and may contribute to immune suppression and promote proliferation and dissemination. These trends persisted at the species level, indicating that they were not driven by association with particularCryptococcusspecies. Isolates possessing giant cells, micro cells, and shed capsule were rare, but strikingly were associated with patient death (p=0.0165). Our data indicate that pleomorphism is an important driver inCryptococcusinfection.ImportanceCryptococcosis results in hundreds of thousands of deaths annually, predominantly in sub-Saharan Africa.Cryptococcusis an encapsulated yeast, and during infection cells have the capacity for substantial morphological changes, including capsule enlargement and shedding, and variations in cell shape and size. In this study we examined 70Cryptococcusisolates causing meningitis in HIV/AIDS patients in Botswana in order to look for associations between phenotypic variation and clinical symptoms. Four variant phenotypes were seen across strains: giant cells ≥ 15 μm, micro cells ≤ 1 μm, shed extracellular capsule, and irregularly shaped cells. We found “large” and “small” phenotypes were associated with differing disease symptoms, indicating that their production may be important during the disease process. Overall, our study indicates thatCryptococcusstrains that can switch on cell types under different situations may be more able to sustain infection and resist the host response.
- Published
- 2018
45. Comparative Population Genomics Analysis of the Mammalian Fungal Pathogen Pneumocystis
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Geetha Kutty, Liang Ma, John P. Dekker, Pavel P. Khil, Joseph A. Kovacs, Xilong Deng, Marco Pagni, Jason E. Stajich, Philippe M. Hauser, Ousmane H. Cissé, Lisa R. Bishop, Richard A. Lempicki, Christina A. Cuomo, Vanessa M. Hirsch, Da-Wei Huang, and Yueqin Liu
- Subjects
0301 basic medicine ,Genetic diversity ,education.field_of_study ,genetic recombination ,Demographic history ,030106 microbiology ,Population ,evolutionary biology ,Genomics ,population structure ,genetic diversity ,Biology ,Microbiology ,QR1-502 ,3. Good health ,Population genomics ,03 medical and health sciences ,030104 developmental biology ,Evolutionary biology ,Virology ,Genetic variation ,pneumonia ,Adaptation ,Pneumocystis Infections ,education - Abstract
Pneumocystis species are opportunistic mammalian pathogens that cause severe pneumonia in immunocompromised individuals. These fungi are highly host specific and uncultivable in vitro . Human Pneumocystis infections present major challenges because of a limited therapeutic arsenal and the rise of drug resistance. To investigate the diversity and demographic history of natural populations of Pneumocystis infecting humans, rats, and mice, we performed whole-genome and large-scale multilocus sequencing of infected tissues collected in various geographic locations. Here, we detected reduced levels of recombination and variations in historical demography, which shape the global population structures. We report estimates of evolutionary rates, levels of genetic diversity, and population sizes. Molecular clock estimates indicate that Pneumocystis species diverged before their hosts, while the asynchronous timing of population declines suggests host shifts. Our results have uncovered complex patterns of genetic variation influenced by multiple factors that shaped the adaptation of Pneumocystis populations during their spread across mammals. IMPORTANCE Understanding how natural pathogen populations evolve and identifying the determinants of genetic variation are central issues in evolutionary biology. Pneumocystis , a fungal pathogen which infects mammals exclusively, provides opportunities to explore these issues. In humans, Pneumocystis can cause a life-threatening pneumonia in immunosuppressed individuals. In analysis of different Pneumocystis species infecting humans, rats, and mice, we found that there are high infection rates and that natural populations maintain a high level of genetic variation despite low levels of recombination. We found no evidence of population structuring by geography. Our comparisons of the times of divergence of these species to their respective hosts suggest that Pneumocystis may have undergone recent host shifts. The results demonstrate that Pneumocystis strains are widely disseminated geographically and provide a new understanding of the evolution of these pathogens.
- Published
- 2018
46. Impact of paracoccin gene silencing on Paracoccidioides brasiliensis Virulence
- Author
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Fabrício F. Fernandes, Aline F. Oliveira, Taise N. Landgraf, Cristina Cunha, Agostinho Carvalho, Patríícia E. Vendruscolo, Relber A. Goncales, Fausto Almeida, Thiago A. da Silva, Fernando Rodrigues, Maria Cristina Roque-Barreira, Christina A. Cuomo, Antonio Cassone, and Universidade do Minho
- Subjects
0301 basic medicine ,Medicina Básica [Ciências Médicas] ,030106 microbiology ,Virulence ,Microbiology ,Fungal virulence ,03 medical and health sciences ,ATMT ,Virology ,Paracoccin silencing ,medicine ,Gene silencing ,2. Zero hunger ,Paracoccidioides brasiliensis ,Science & Technology ,biology ,Paracoccin ,Paracoccidioidomycosis ,Chitinase ,biology.organism_classification ,medicine.disease ,QR1-502 ,SURTOS DE DOENÇAS ,Ciências Médicas::Medicina Básica ,Christian ministry - Abstract
Among the endemic deep mycoses in Latin America, paracoccidioidomycosis (PCM), caused by thermodimorphic fungi of the Paracoccidioides genus, is a major cause of morbidity. Disease development and its manifestations are associated with both host and fungal factors. Concerning the latter, several recent studies have employed the methodology of gene modulation in P. brasiliensis using antisense RNA (AsRNA) and Agrobacterium tumefaciens-mediated transformation (ATMT) to identify proteins that influence fungus virulence. Our previous observations suggested that paracoccin (PCN), a multidomain fungal protein with both lectin and enzymatic activities, may be a potential P. brasiliensis virulence factor. To explore this, we used AsRNA and ATMT methodology to obtain three independent PCN-silenced P. brasiliensis yeast strains (AsPCN1, AsPCN2, and AsPCN3) and characterized them with regard to P. brasiliensis biology and pathogenicity. AsPCN1, AsPCN2, and AsPCN3 showed relative PCN expression levels that were 60%, 40%, and 60% of that of the wild-type (WT) strain, respectively. PCN silencing led to the aggregation of fungal cells, blocked the morphological yeast-to-mycelium transition, and rendered the yeast less resistant to macrophage fungicidal activity. In addition, mice infected with AsPCN1, AsPCN2, and AsPCN3 showed a reduction in fungal burden of approximately 96% compared with those inoculated with the WT strain, which displayed a more extensive destruction of lung tissue. Finally, mice infected with the PCN-silenced yeast strains had lower mortality than those infected with the WT strain. These data demonstrate that PCN acts as a P. brasiliensis contributory virulence factor directly affecting fungal pathogenesis. IMPORTANCE The nonexistence of efficient genetic transformation systems has hampered studies in the dimorphic fungus Paracoccidioides brasiliensis, the etiological agent of the most frequent systemic mycosis in Latin America. The recent development of a method for gene expression knockdown by antisense RNA technology, associated with an Agrobacterium tumefaciens-mediated transformation system, provides new strategies for studying P. brasiliensis. Through this technology, we generated yeasts that were silenced for paracoccin (PCN), a P. brasiliensis component that has lectin and enzymatic properties. By comparing the phenotypes of PCN-silenced and wild-type strains of P. brasiliensis, we identified PCN as a virulence factor whose absence renders the yeasts unable to undergo the transition to mycelium and causes a milder pulmonary disease in mice, with a lower mortality rate. Our report highlights the importance of the technology used for P. brasiliensis transformation and demonstrates that paracoccin is a virulence factor acting on fungal biology and pathogenesis., This work had financial support from the following agencies: Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (2016/00629-4, 2014/05359-0, 2012/08552-0, 2014/22561-7, 2012/09611-0, 2016/04877-2, 2016/60642-2, and 2013/04088-0); Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (150036/2014-0, 477161/2008-1, and 475357/2013-2); Financiadora de Estudos e Projetos – FINEP (0110045900); Ministry of Education and Science – Fundação para a Ciência e a Tecnologia – FCT (SFRH/BPD/96176/2013 and IF/00735/2014), info:eu-repo/semantics/publishedVersion
- Published
- 2017
47. Microevolution of Serial Clinical Isolates of C. gattii
- Author
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Yuan Chen, Rhys A. Farrer, Charles Giamberardino, Sharadha Sakthikumar, Alexander Jones, Timothy Yang, Jennifer L. Tenor, Omar Wagih, Marelize Van Wyk, Nelesh P. Govender, Thomas G. Mitchell, Anastasia P. Litvintseva, Christina A. Cuomo, John R. Perfect, and Francoise Dromer
- Subjects
0301 basic medicine ,Genotype ,Cryptococcus ,Antifungal drug ,Adaptation, Biological ,Virulence ,Drug resistance ,Meningitis, Cryptococcal ,Microbiology ,Evolution, Molecular ,03 medical and health sciences ,South Africa ,Drug Resistance, Fungal ,Recurrence ,Virology ,medicine ,Humans ,Longitudinal Studies ,Cryptococcus gattii ,Cerebrospinal Fluid ,Cryptococcus neoformans ,biology ,Temperature ,biology.organism_classification ,QR1-502 ,3. Good health ,030104 developmental biology ,Phenotype ,Fluconazole ,medicine.drug ,Research Article - Abstract
The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis., IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.
- Published
- 2017
48. Analysis of a food-borne fungal pathogen outbreak: virulence and genome of a Mucor circinelloides isolate from yogurt
- Author
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Eun Young Huh, Sean M. Sykes, Soo Chan Lee, Christina A. Cuomo, Alicia Li, Joseph Heitman, Sandra Carson, Piotr A. Mieczkowski, R. Blake Billmyre, and Dennis C. Ko
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Male ,Mating type ,Virulence ,Biology ,Microbiology ,Disease Outbreaks ,Mice ,Virology ,Animals ,Humans ,Mucormycosis ,Typing ,Feces ,2. Zero hunger ,Mucor ,Mice, Inbred BALB C ,Outbreak ,food and beverages ,biology.organism_classification ,Yogurt ,QR1-502 ,3. Good health ,Mucor circinelloides ,Genome, Fungal ,Bacteria ,Research Article - Abstract
Food-borne pathogens are ongoing problems, and new pathogens are emerging. The impact of fungi, however, is largely underestimated. Recently, commercial yogurts contaminated with Mucor circinelloides were sold, and >200 consumers became ill with nausea, vomiting, and diarrhea. Mucoralean fungi cause the fatal fungal infection mucormycosis, whose incidence has been continuously increasing. In this study, we isolated an M. circinelloides strain from a yogurt container, and multilocus sequence typing identified the strain as Mucor circinelloides f. circinelloides. M. circinelloides f. circinelloides is the most virulent M. circinelloides subspecies and is commonly associated with human infections, whereas M. circinelloides f. lusitanicus and M. circinelloides f. griseocyanus are less common causes of infection. Whole-genome analysis of the yogurt isolate confirmed it as being close to the M. circinelloides f. circinelloides subgroup, with a higher percentage of divergence with the M. circinelloides f. lusitanicus subgroup. In mating assays, the yogurt isolate formed sexual zygospores with the (−) M. circinelloides f. circinelloides tester strain, which is congruent with its sex locus encoding SexP, the (+) mating type sex determinant. The yogurt isolate was virulent in murine and wax moth larva host systems. In a murine gastromucormycosis model, Mucor was recovered from fecal samples of infected mice for up to 10 days, indicating that Mucor can survive transit through the GI tract. In interactions with human immune cells, M. circinelloides f. lusitanicus induced proinflammatory cytokines but M. circinelloides f. circinelloides did not, which may explain the different levels of virulence in mammalian hosts. This study demonstrates that M. circinelloides can spoil food products and cause gastrointestinal illness in consumers and may pose a particular risk to immunocompromised patients., IMPORTANCE The U.S. FDA reported that yogurt products were contaminated with M. circinelloides, a mucoralean fungal pathogen, and >200 consumers complained of symptoms, including vomiting, nausea, and diarrhea. The manufacturer voluntarily withdrew the affected yogurt products from the market. Compared to other food-borne pathogens, including bacteria, viruses, and parasites, less focus has been placed on the risk of fungal pathogens. This study evaluates the potential risk from the food-borne fungal pathogen M. circinelloides that was isolated from the contaminated commercial yogurt. We successfully cultured an M. circinelloides isolate and found that the isolate belongs to the species M. circinelloides f. circinelloides, which is often associated with human infections. In murine and insect host models, the isolate was virulent. While information disseminated in the popular press would suggest this fungal contaminant poses little or no risk to consumers, our results show instead that it is capable of causing significant infections in animals.
- Published
- 2014
49. Highly recombinant VGII Cryptococcus gattii population develops clonal outbreak clusters through both sexual macroevolution and asexual microevolution
- Author
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James W. Kronstad, Piotr A. Mieczkowski, Dee A. Carter, Wenjun Li, Christina A. Cuomo, Joseph Heitman, Daniel Croll, and R. Blake Billmyre
- Subjects
Genetics ,education.field_of_study ,Virulence ,biology ,Population ,Introgression ,Outbreak ,Microevolution ,Cryptococcus gattii ,biology.organism_classification ,Microbiology ,Linkage Disequilibrium ,QR1-502 ,Disease Outbreaks ,3. Good health ,Sexual reproduction ,Haplotypes ,Virology ,education ,Clade ,Research Article - Abstract
An outbreak of the fungal pathogen Cryptococcus gattii began in the Pacific Northwest (PNW) in the late 1990s. This outbreak consists of three clonal subpopulations: VGIIa/major, VGIIb/minor, and VGIIc/novel. Both VGIIa and VGIIc are unique to the PNW and exhibit increased virulence. In this study, we sequenced the genomes of isolates from these three groups, as well as global isolates, and analyzed a total of 53 isolates. We found that VGIIa/b/c populations show evidence of clonal expansion in the PNW. Whole-genome sequencing provided evidence that VGIIb originated in Australia, while VGIIa may have originated in South America, and these were likely independently introduced. Additionally, the VGIIa outbreak lineage may have arisen from a less virulent clade that contained a mutation in the MSH2 ortholog, but this appears to have reverted in the VGIIa outbreak strains, suggesting that a transient mutator phenotype may have contributed to adaptation and evolution of virulence in the PNW outbreak. PNW outbreak isolates share genomic islands, both between the clonal lineages and with global isolates, indicative of sexual recombination. This suggests that VGII C. gattii has undergone sexual reproduction, either bisexual or unisexual, in multiple locales contributing to the production of novel, virulent subtypes. We also found that the genomes of two basal VGII isolates from HIV+ patients contain an introgression tract spanning three genes. Introgression substantially contributed to intra-VGII polymorphism and likely occurred through sexual reproduction with VGI. More broadly, these findings illustrate how both microevolution and sexual reproduction play central roles in the development of infectious outbreaks from avirulent or less virulent progenitors., IMPORTANCE Cryptococcus gattii is the causative agent responsible for ongoing infections in the Pacific Northwest of the United States and western Canada. The incidence of these infections increased dramatically in the 1990s and remains elevated. These infections are attributable to three clonal lineages of C. gattii, VGIIa, VGIIb, and VGIIc, with only VGIIa identified once previously in the Pacific Northwest prior to the start of the outbreak, albeit in a less virulent form. This study addresses the origin and emergence of this outbreak, using whole-genome sequencing and comparison of both outbreak and global isolates. We show that VGIIa arose mitotically from a less virulent clonal group, possibly via the action of a mutator phenotype, while VGIIb was likely introduced from Australia, and VGIIc appears to have emerged in the United States or in an undersampled locale via sexual reproduction. This work shows that multiple processes can contribute to the emergence of an outbreak.
- Published
- 2014
50. Comparative Genome Analysis of Trichophyton rubrum and Related Dermatophytes Reveals Candidate Genes Involved in Infection
- Author
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Zehua Chen, Sakina Saif, Wenjun Li, Terrance Shea, Sarah J. Gurr, Ekaterina Shelest, Elizabeth Birch, Charles Winston Saunders, Axel A. Brakhage, Jun Xu, David I. Heiman, Michel Monod, Idit Kosti, Qiandong Zeng, Diego Martinez, Christina A. Cuomo, Sarah Young, Theodore C. White, Jonathan M. Goldberg, Brian G. Oliver, Richard C. Barton, Marketa Samalova, Yvonne Gräser, Richard C. Summerbell, Joseph Heitman, Antonio Rossi, Bruce W. Birren, and Nilce Maria Martinez-Rossi
- Subjects
Virulence Factors ,Genes, Fungal ,Molecular Sequence Data ,Trichophyton rubrum ,medicine.disease_cause ,Genome ,Microbiology ,03 medical and health sciences ,Trichophyton ,Virology ,medicine ,Gene family ,Humans ,Microsporum ,DNA, Fungal ,Gene ,Trichophyton tonsurans ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,Arthrodermataceae ,FUNGOS ,Sequence Analysis, DNA ,biology.organism_classification ,QR1-502 ,Dermatophyte ,Genome, Fungal ,Research Article - Abstract
The major cause of athlete’s foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response., IMPORTANCE Athlete’s foot, jock itch, ringworm, and nail infections are common fungal infections, all caused by fungi known as dermatophytes (fungi that infect skin). This report presents the genome sequences of Trichophyton rubrum, the most frequent cause of athlete’s foot, as well as four other common dermatophytes. Dermatophyte genomes are enriched for four gene classes that may contribute to the ability of these fungi to cause disease. These include (i) proteases secreted to degrade skin; (ii) kinases, including pseudokinases, that are involved in signaling necessary for adapting to skin; (iii) secondary metabolites, compounds that act as toxins or signals in the interactions between fungus and host; and (iv) a class of proteins (LysM) that appear to bind and mask cell wall components and carbohydrates, thus avoiding the host’s immune response to the fungi. These genome sequences provide a strong foundation for future work in understanding how dermatophytes cause disease.
- Published
- 2012
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