Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

The genome of Naegleria gruberi illuminates early eukaryotic versatility.

Genome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems.

Isolation and maintenance of Batrachochytrium salamandrivorans cultures.

Discovered in 2013, the chytrid fungus Batrachochytrium salamandrivorans (Bsal) is an emerging amphibian pathogen that causes ulcerative skin lesions and multifocal erosion. A closely related pathogen, B. dendrobatidis (Bd), has devastated amphibian populations worldwide, suggesting that Bsal poses a significant threat to global salamander biodiversity. To expedite research into this emerging threat, we seek to standardize protocols across the field so that results of laboratory studies are reproducible and comparable. We have collated data and experience from multiple labs to standardize culturing practices of Bsal. Here we outline common culture practices including a medium for standardized Bsal growth, standard culturing protocols, and a method for isolating Bsal from infected tissue.

Non-model model organisms.

Model organisms are widely used in research as accessible and convenient systems to study a particular area or question in biology. Traditionally only a handful of organisms have been widely studied, but modern research tools are enabling researchers to extend the set of model organisms to include less-studied and more unusual systems. This Forum highlights a range of 'non-model model organisms' as emerging systems for tackling questions across the whole spectrum of biology (and beyond), the opportunities and challenges, and the outlook for the future.

An internally controlled system to study microtubule network diversification links tubulin evolution to the use of distinct microtubule regulators.

Diverse eukaryotic cells assemble microtubule networks that vary in structure and composition. While we understand how cells build microtubule networks with specialized functions, we do not know how microtubule networks diversify across deep evolutionary timescales. This problem has remained unresolved because most organisms use shared pools of tubulins for multiple networks, making it impossible to trace the evolution of any single network. In contrast, the amoeboflagellate Naegleria uses distinct tubulin genes to build distinct microtubule networks: while Naegleria builds flagella from conserved tubulins during differentiation, it uses divergent tubulins to build its mitotic spindle. This genetic separation makes for an internally controlled system to study independent microtubule networks in a single organismal and genomic context. To explore the evolution of these microtubule networks, we identified conserved microtubule binding proteins and used transcriptional profiling of mitosis and differentiation to determine which are upregulated during the assembly of each network. Surprisingly, most microtubule binding proteins are upregulated during only one process, suggesting that Naegleria uses distinct component pools to specialize its microtubule networks. Furthermore, the divergent residues of mitotic tubulins tend to fall within the binding sites of differentiation-specific microtubule regulators, suggesting that interactions between microtubules and their binding proteins constrain tubulin sequence diversification. We therefore propose a model for cytoskeletal evolution in which pools of microtubule network components constrain and guide the diversification of the entire network, so that the evolution of tubulin is inextricably linked to that of its binding partners.

An endogenous DNA virus in an amphibian-killing fungus associated with pathogen genotype and virulence.

The global panzootic lineage (GPL) of the pathogenic fungus Batrachochytrium dendrobatidis (Bd) has caused severe amphibian population declines, yet the drivers underlying the high frequency of GPL in regions of amphibian decline are unclear. Using publicly available Bd genome sequences, we identified multiple non-GPL Bd isolates that contain a circular Rep-encoding single-stranded (CRESS)-like DNA virus, which we named Bd DNA virus 1 (BdDV-1). We further sequenced and constructed genome assemblies with long read sequences to find that the virus is integrated into the nuclear genome in some strains. Attempts to cure virus-positive isolates were unsuccessful; however, phenotypic differences between naturally virus-positive and virus-negative Bd isolates suggested that BdDV-1 decreases the growth of its host in vitro but increases the virulence of its host in vivo. BdDV-1 is the first-described CRESS DNA mycovirus of zoosporic true fungi, with a distribution inversely associated with the emergence of the panzootic lineage.

Plant-type mitochondrial RNA editing in the protist Naegleria gruberi.

RNA editing converts hundreds of cytidines into uridines in plant mitochondrial and chloroplast transcripts. Recognition of the RNA editing sites in the organelle transcriptomes requires numerous specific, nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins with characteristic carboxy-terminal protein domain extensions (E/DYW) previously thought to be unique to plants. However, a small gene family of such plant-like PPR proteins of the DYW-type was recently discovered in the genome of the protist Naegleria gruberi. This raised the possibility that plant-like RNA editing may occur in this amoeboflagellate. Accordingly, we have investigated the mitochondrial transcriptome of Naegleria gruberi and here report on identification of two sites of C-to-U RNA editing in the cox1 gene and in the cox3 gene, both of which reconstitute amino acid codon identities highly conserved in evolution. An estimated 1.5 billion years of evolution separate the heterolobosean protist Naegleria from the plant lineage. The new findings either suggest horizontal gene transfer of RNA editing factors or that plant-type RNA editing is evolutionarily much more ancestral than previously thought and yet to be discovered in many other ancient eukaryotic lineages.

The evolution and diversity of actin-dependent cell migration.

Many eukaryotic cells, including animal cells and unicellular amoebae, use dynamic-actin networks to crawl across solid surfaces. Recent discoveries of actin-dependent crawling in additional lineages have sparked interest in understanding how and when this type of motility evolved. Tracing the evolution of cell crawling requires understanding the molecular mechanisms underlying motility. Here we outline what is known about the diversity and evolution of the molecular mechanisms that drive cell motility, with a focus on actin-dependent crawling. Classic studies and recent work have revealed a surprising number of distinct mechanical modes of actin-dependent crawling used by different cell types and species to navigate different environments. The overlap in actin network regulators driving multiple types of actin-dependent crawling, along with cortical-actin networks that support the plasma membrane in these cells, suggest that actin motility and cortical actin networks might have a common evolutionary origin. The rapid development of additional evolutionarily diverse model systems, advanced imaging technologies, and CRISPR-based genetic tools, is opening the door to testing these and other new ideas about the evolution of actin-dependent cell crawling.

Evolutionary cell biology: New roles for Arp2/3 complex evolution in eukaryotic diversification.

The actin cytoskeleton is a protein polymer system that underlies a wide variety of eukaryotic phenotypes. A new study reports that diversity in a key actin regulator, the Arp2/3 complex, drives species-specific sperm development within the Drosophila lineage.

A guide to Agrobacterium-mediated transformation of the chytrid fungus Spizellomyces punctatus.

Chytrid fungi play key ecological roles in aquatic ecosystems, and some species cause a devastating skin disease in frogs and salamanders. Additionally, chytrids occupy a unique phylogenetic position- sister to the well-studied Dikarya (the group including yeasts, sac fungi, and mushrooms) and related to animals- making chytrids useful for answering important evolutionary questions. Despite their importance, little is known about the basic cell biology of chytrids. A major barrier to understanding chytrid biology has been a lack of genetic tools with which to test molecular hypotheses. Medina and colleagues recently developed a protocol for Agrobacterium -mediated transformation of Spizellomyces punctatus. In this manuscript, we describe the general procedure including planning steps and expected results. We also provide in-depth, step-by-step protocols and video guides for performing the entirety of this transformation procedure on protocols.io (dx.doi.org/10.17504/protocols.io.x54v9dd1pg3e/v1).

A conserved pressure-driven mechanism for regulating cytosolic osmolarity.

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

A conserved pressure-driven mechanism for regulating cytosolic osmolarity.

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineagesâ€"the discoban Naegleria gruberi , and the amoebozoan slime mold Dictyostelium discoideum . Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum . Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

Ancestral centriole and flagella proteins identified by analysis of Naegleria differentiation.

Naegleria gruberi is a single-celled eukaryote best known for its remarkable ability to form an entire microtubule cytoskeleton de novo during its metamorphosis from an amoeba into a flagellate, including basal bodies (equivalent to centrioles), flagella and a cytoplasmic microtubule array. Our publicly available full-genome transcriptional analysis, performed at 20-minute intervals throughout Naegleria differentiation, reveals vast transcriptional changes, including the differential expression of genes involved in metabolism, signaling and the stress response. Cluster analysis of the transcriptional profiles of predicted cytoskeletal genes reveals a set of 55 genes enriched in centriole components (induced early) and a set of 82 genes enriched in flagella proteins (induced late). The early set includes genes encoding nearly every known conserved centriole component, as well as eight previously uncharacterized, highly conserved genes. The human orthologs of at least five genes localize to the centrosomes of human cells, one of which (here named Friggin) localizes specifically to mother centrioles.

Amphibian mucus triggers a developmental transition in the frog-killing chytrid fungus.

The frog-killing chytrid fungus Batrachochytrium dendrobatidis (Bd) is decimating amphibian populations around the world.1-4Bd has a biphasic life cycle, alternating between motile zoospores that disperse within aquatic environments and sessile sporangia that grow within the mucus-coated skin of amphibians.5,6 Zoospores lack cell walls and swim rapidly through aquatic environments using a posterior flagellum and crawl across solid surfaces using actin structures similar to those of human cells.7,8Bd transitions from this motile dispersal form to its reproductive form by absorbing its flagellum, rearranging its actin cytoskeleton, and rapidly building a chitin-based cell wall-a process called "encystation."5-7 The resulting sporangium increases in volume by two or three orders of magnitude while undergoing rounds of mitosis without cytokinesis to form a large ceonocyte. The sporangium then cellurizes by dividing its cytoplasm into dozens of new zoospores. After exiting the sporangium through a discharge tube onto the amphibian skin, daughter zoospores can then reinfect the same individual or find a new host.5 Although encystation is critical to Bd growth, whether and how this developmental transition is triggered by external signals was previously unknown. We discovered that exposure to amphibian mucus triggers rapid and reproducible encystation within minutes. This response can be recapitulated with purified mucin, the bulk component of mucus, but not by similarly viscous methylcellulose or simple sugars. Mucin-induced encystation does not require gene expression but does require surface adhesion, calcium signaling, and modulation of the actin cytoskeleton. Mucus-induced encystation may represent a key mechanism for synchronizing Bd development with the arrival at the host.

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.

The future of fungi: threats and opportunities.

The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Naegleria gruberi de novo basal body assembly occurs via stepwise incorporation of conserved proteins.

Centrioles and basal bodies are discrete structures composed of a cylinder of nine microtubule triplets and associated proteins. Metazoan centrioles can be found at mitotic spindle poles and are called basal bodies when used to organize microtubules to form the core structure of flagella. Naegleria gruberi, a unicellular eukaryote, grows as an amoeba that lacks a cytoplasmic microtubule cytoskeleton. When stressed, Naegleria rapidly (and synchronously) differentiates into a flagellate, forming a complete cytoplasmic cytoskeleton de novo, including two basal bodies and flagella. Here, we show that Naegleria has genes encoding conserved centriole proteins. Using novel antibodies, we describe the localization of three centrosomal protein homologs (SAS-6, gamma-tubulin, and centrin-1) during the assembly of the flagellate microtubule cytoskeleton. We also used these antibodies to show that Naegleria expresses the proteins in the same order as their incorporation into basal bodies, with SAS-6 localizing first, followed by centrin and finally gamma-tubulin. The similarities between basal body assembly in Naegleria and centriole assembly in animals indicate that mechanisms of assembly, as well as structure, have been conserved throughout eukaryotic evolution.