During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.

Candida albicans is the most common cause of fungal infection in humans. IL-17 is critical for defense against superficial fungal infections, but the role of this response in invasive disease is less understood. We show that C. albicans secretes a lipase, Lip2, that facilitates invasive disease via lipid-based suppression of the IL-17 response. Lip2 was identified as an essential virulence factor in a forward genetic screen in a mouse model of bloodstream infection. Murine infection with C. albicans strains lacking Lip2 display exaggerated IL-17 responses that lead to fungal clearance from solid organs and host survival. Both IL-17 signaling and lipase activity are required for Lip2-mediated suppression. Lip2 inhibits IL-17 production indirectly by suppressing IL-23 production by tissue-resident dendritic cells. The lipase hydrolysis product, palmitic acid, similarly suppresses dendritic cell activation in vitro. Thus, C. albicans suppresses antifungal IL-17 defense in solid organs by altering the tissue lipid milieu.

Understanding the molecular mechanisms of pathogen emergence is central to mitigating the impacts of novel infectious disease agents. The chytrid fungus Batrachochytrium dendrobatidis (Bd) is an emerging pathogen of amphibians that has been implicated in amphibian declines worldwide. Bd is the only member of its clade known to attack vertebrates. However, little is known about the molecular determinants of - or evolutionary transition to - pathogenicity in Bd. Here we sequence the genome of Bd's closest known relative - a non-pathogenic chytrid Homolaphlyctis polyrhiza (Hp). We first describe the genome of Hp, which is comparable to other chytrid genomes in size and number of predicted proteins. We then compare the genomes of Hp, Bd, and 19 additional fungal genomes to identify unique or recent evolutionary elements in the Bd genome. We identified 1,974 Bd-specific genes, a gene set that is enriched for protease, lipase, and microbial effector Gene Ontology terms. We describe significant lineage-specific expansions in three Bd protease families (metallo-, serine-type, and aspartyl proteases). We show that these protease gene family expansions occurred after the divergence of Bd and Hp from their common ancestor and thus are localized to the Bd branch. Finally, we demonstrate that the timing of the protease gene family expansions predates the emergence of Bd as a globally important amphibian pathogen.

Filamentous fungi undergo somatic cell fusion to create a syncytial, interconnected hyphal network which confers a fitness benefit during colony establishment. However, barriers to somatic cell fusion between genetically different cells have evolved that reduce invasion by parasites or exploitation by maladapted genetic entities (cheaters). Here, we identified a predicted mannosyltransferase, glycosyltransferase family 69 protein (GT69-2) that was required for somatic cell fusion in Neurospora crassa Cells lacking GT69-2 prematurely ceased chemotropic signaling and failed to complete cell wall dissolution and membrane merger in pairings with wild-type cells or between Δgt69-2 cells (self fusion). However, loss-of-function mutations in the linked regulator of cell fusion and cell wall remodeling-1 (rfw-1) locus suppressed the self-cell-fusion defects of Δgt69-2 cells, although Δgt69-2 Δrfw-1 double mutants still failed to undergo fusion with wild-type cells. Both GT69-2 and RFW-1 localized to the Golgi apparatus. Genetic analyses indicated that RFW-1 negatively regulates cell wall remodeling-dependent processes, including cell wall dissolution during cell fusion, separation of conidia during asexual sporulation, and conidial germination. GT69-2 acts as an antagonizer to relieve or prevent negative functions on cell fusion by RFW-1. In Neurospora species and N. crassa populations, alleles of gt69-2 were highly polymorphic and fell into two discrete haplogroups. In all isolates within haplogroup I, rfw-1 was conserved and linked to gt69-2 All isolates within haplogroup II lacked rfw-1. These data indicated that gt69-2/rfw-1 are under balancing selection and provide new mechanisms regulating cell wall remodeling during cell fusion and conidial separation.IMPORTANCE Cell wall remodeling is a dynamic process that balances cell wall integrity versus cell wall dissolution. In filamentous fungi, cell wall dissolution is required for somatic cell fusion and conidial separation during asexual sporulation. In the filamentous fungus Neurospora crassa, allorecognition checkpoints regulate the cell fusion process between genetically different cells. Our study revealed two linked loci with transspecies polymorphisms and under coevolution, rfw-1 and gt69-2, which form a coordinated system to regulate cell wall remodeling during somatic cell fusion, conidial separation, and asexual spore germination. RFW-1 acts as a negative regulator of these three processes, while GT69-2 functions antagonistically to RFW-1. Our findings provide new insight into the mechanisms involved in regulation of fungal cell wall remodeling during growth and development.

"Fungi on Mars!": a popular news heading that piques public interest and makes scientists' blood boil. While such a statement is laden with misinformation and light on evidence, the search for past and present extraterrestrial life is an ongoing scientific effort. Moreover, it is one that is increasingly gaining momentum with the recent collection of martian rock cores from Jezero Crater by NASA's Perseverance rover. Despite the increasingly sophisticated approaches guiding the search for microbial life on other planets, fungi remain relatively underexplored compared to their bacterial counterparts, highlighting a gap between the astrobiological and fungal research communities. Through a meeting in April 2021, the CIFAR Earth 4D and Fungal Kingdom research programs worked to bridge this divide by uniting experts in each field. CIFAR is a Canadian-based global research organization that convenes researchers across disciplines to address important questions facing science and humanity. The CIFAR Earth 4D: Subsurface Science & Exploration and Fungal Kingdom: Threats & Opportunities research programs were launched by CIFAR in July 2019, each made up of approximately two dozen international researchers who are experts in their fields. The Earth 4D program, led by co-directors John Mustard (Brown University, USA) and Barbara Sherwood Lollar (University of Toronto, Canada), aims to understand the complex chemical, physical, and biological interactions that occur within and between Earth's surface and subsurface to explore questions on the evolution of planets and life. The Fungal Kingdom program, led by co-directors Leah Cowen (University of Toronto, Canada) and Joseph Heitman (Duke University, USA), seeks to tackle the most pressing threats fungi pose to human health, agriculture, and biodiversity and to harness their extraordinary potential. The programs met to explore areas for synergy within four major themes: (1) the origins of life; (2) the evolution and diversification of life; (3) life in diverse and extreme environments; and (4) extinction: lessons learned and threats. This report covers the research discussed during the meeting across these four themes.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

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.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn₂C₆ transcription factors YRR1 and YRM1 (p < 1 × 10^-100). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency.

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.

Unlike superficial fungal infections of the skin and nails, which are the most common fungal diseases in humans, invasive fungal infections carry high morbidity and mortality, particularly those associated with biofilm formation on indwelling medical devices. Therapeutic management of these complex diseases is often complicated by the rise in resistance to the commonly used antifungal agents. Therefore, the availability of accurate susceptibility testing methods for determining antifungal resistance, as well as discovery of novel antifungal and antibiofilm agents, are key priorities in medical mycology research. To direct advancements in this field, here we present an overview of the methods currently available for determining (i) the susceptibility or resistance of fungal isolates or biofilms to antifungal or antibiofilm compounds and compound combinations; (ii) the in vivo efficacy of antifungal and antibiofilm compounds and compound combinations; and (iii) the in vitro and in vivo performance of anti-infective coatings and materials to prevent fungal biofilm-based infections.