The actin networks of chytrid fungi reveal evolutionary loss of cytoskeletal complexity in the fungal kingdowm

Cells from across the eukaryotic tree use actin polymer networks for a wide variety of functions, including endocytosis, cytokinesis, and cell migration. Despite this functional conservation, the actin cytoskeleton has undergone significant diversification, highlighted by the differences in the actin networks of mammalian cells and yeast. Chytrid fungi diverged before the emergence of the Dikarya (multicellular fungi and yeast) and therefore provide a unique opportunity to study actin cytoskeletal evolution. Chytrids have two life stages: zoospore cells that can swim with a flagellum and sessile sporangial cells that, like multicellular fungi, are encased in a chitinous cell wall. Here, we show that zoospores of the amphibian-killing chytrid Batrachochytrium dendrobatidis (Bd) build dynamic actin structures resembling those of animal cells, including an actin cortex, pseudopods, and filopodia-like spikes. In contrast, Bd sporangia assemble perinuclear actin shells and actin patches similar to those of yeast. The use of specific small-molecule inhibitors indicate that nearly all of Bd’s actin structures are dynamic and use distinct nucleators: although pseudopods and actin patches are Arp2/3 dependent, the actin cortex appears formin dependent and actin spikes require both nucleators. Our analysis of multiple chytrid genomes reveals actin regulators and myosin motors found in animals, but not dikaryotic fungi, as well as fungal-specific components. The presence of animal- and yeast-like actin cytoskeletal components in the genome combined with the intermediate actin phenotypes in Bd suggests that the simplicity of the yeast cytoskeleton may be due to evolutionary loss

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

A nonmitochondrial hydrogen production in Naegleria gruberi

Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes

The Structure of the Mitotic Spindle and Nucleolus during Mitosis in the Amebo-Flagellate Naegleria

Mitosis in the amebo-flagellate Naegleria pringsheimi is acentrosomal and closed (the nuclear membrane does not break down). The large central nucleolus, which occupies about 20% of the nuclear volume, persists throughout the cell cycle. At mitosis, the nucleolus divides and moves to the poles in association with the chromosomes. The structure of the mitotic spindle and its relationship to the nucleolus are unknown. To identify the origin and structure of the mitotic spindle, its relationship to the nucleolus and to further understand the influence of persistent nucleoli on cellular division in acentriolar organisms like Naegleria, three-dimensional reconstructions of the mitotic spindle and nucleolus were carried out using confocal microscopy. Monoclonal antibodies against three different nucleolar regions and α-tubulin were used to image the nucleolus and mitotic spindle. Microtubules were restricted to the nucleolus beginning with the earliest prophase spindle microtubules. Early spindle microtubules were seen as short rods on the surface of the nucleolus. Elongation of the spindle microtubules resulted in a rough cage of microtubules surrounding the nucleolus. At metaphase, the mitotic spindle formed a broad band completely embedded within the nucleolus. The nucleolus separated into two discreet masses connected by a dense band of microtubules as the spindle elongated. At telophase, the distal ends of the mitotic spindle were still completely embedded within the daughter nucleoli. Pixel by pixel comparison of tubulin and nucleolar protein fluorescence showed 70% or more of tubulin co-localized with nucleolar proteins by early prophase. These observations suggest a model in which specific nucleolar binding sites for microtubules allow mitotic spindle formation and attachment. The fact that a significant mass of nucleolar material precedes the chromosomes as the mitotic spindle elongates suggests that spindle elongation drives nucleolar division

The Genome Portal of the Department of Energy Joint Genome Institute

The Department of Energy (DOE) Joint Genome Institute (JGI) is a national user facility with massive-scale DNA sequencing and analysis capabilities dedicated to advancing genomics for bioenergy and environmental applications. Beyond generating tens of trillions of DNA bases annually, the Institute develops and maintains data management systems and specialized analytical capabilities to manage and interpret complex genomic data sets, and to enable an expanding community of users around the world to analyze these data in different contexts over the web. The JGI Genome Portal (http://genome.jgi.doe.gov) provides a unified access point to all JGI genomic databases and analytical tools. A user can find all DOE JGI sequencing projects and their status, search for and download assemblies and annotations of sequenced genomes, and interactively explore those genomes and compare them with other sequenced microbes, fungi, plants or metagenomes using specialized systems tailored to each particular class of organisms. We describe here the general organization of the Genome Portal and the most recent addition, MycoCosm (http://jgi.doe.gov/fungi), a new integrated fungal genomics resource

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.publishedVersio

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