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[...]over the course of hours, the transcriptional up-regulation of cytokines and chemokines, including TNF-α and interleukin-4, can be observed. Importantly, each of these responses may occur alone or in combination depending on the stimulus. Because of their location, their plasticity, and the various mediators they produce, mast cells are important immune effector and modulatory cells that help link innate and adaptive immunity in the fight against pathogens.

Invasive fungal infections pose an important threat to public health and are an under-recognized component of antimicrobial resistance, an emerging crisis worldwide. Across a period of profound global environmental change and expanding at-risk populations, human-infecting pathogenic fungi are evolving resistance to all licensed systemic antifungal drugs. In this Review, we highlight the main mechanisms of antifungal resistance and explore the similarities and differences between bacterial and fungal resistance to antimicrobial control. We discuss the research and innovation topics that are needed for risk reduction strategies aimed at minimizing the emergence of resistance in pathogenic fungi. These topics include links between the environment and One Health, surveillance, diagnostics, routes of transmission, novel therapeutics and methods to mitigate hotspots for fungal adaptation. We emphasize the global efforts required to steward our existing antifungal armamentarium, and to direct the research and development of future therapies and interventions.The impacts of fungal infections on human health are of increasing concern, and resistance of pathogenic fungi to all licensed systemic antifungals has been documented. In this Review, Fisher, Verweij and colleagues discuss the research and innovation topics that are needed to understand and minimize the occurrence and impact of antifungal resistance.

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.

Of the over 250 Aspergillus species, Aspergillus fumigatus accounts for up to 80% of invasive human infections. A. fumigatus produces galactosaminogalactan (GAG), an exopolysaccharide composed of galactose and N-acetyl-galactosamine (GalNAc) that mediates adherence and is required for full virulence. Less pathogenic Aspergillus species were found to produce GAG with a lower GalNAc content than A. fumigatus and expressed minimal amounts of cell wall-bound GAG. Increasing the GalNAc content of GAG of the minimally pathogenic A. nidulans, either through overexpression of the A. nidulans epimerase UgeB or by heterologous expression of the A. fumigatus epimerase Uge3 increased the amount of cell wall bound GAG, augmented adherence in vitro and enhanced virulence in corticosteroid-treated mice to levels similar to A. fumigatus. The enhanced virulence of the overexpression strain of A. nidulans was associated with increased resistance to NADPH oxidase-dependent neutrophil extracellular traps (NETs) in vitro, and was not observed in neutropenic mice or mice deficient in NADPH-oxidase that are unable to form NETs. Collectively, these data suggest that cell wall-bound GAG enhances virulence through mediating resistance to NETs.

Fungal biofilms are communities of adherent cells surrounded by an extracellular matrix. These biofilms are commonly found during infection caused by a variety of fungal pathogens. Clinically, biofilm infections can be extremely difficult to eradicate due to their resistance to antifungals and host defenses. Biofilm formation can protect fungal pathogens from many aspects of the innate immune system, including killing by neutrophils and monocytes. Altered immune recognition during this phase of growth is also evident by changes in the cytokine profiles of monocytes and macrophages exposed to biofilm. In this manuscript, we review the host response to fungal biofilms, focusing on how these structures are recognized by the innate immune system. Biofilms formed by , and have received the most attention and are highlighted. We describe common themes involved in the resilience of fungal biofilms to host immunity and give examples of biofilm defenses that are pathogen-specific.

About the Authors: Hanna Ostapska Affiliations Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada, Infectious Diseases in Global Health Program, McGill University Health Centre, Montreal, Quebec, Canada P. Lynne Howell * E-mail: howell@sickkids.ca (PLH); donald.sheppard@mcgill.ca (DCS) Affiliations Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada, Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada ORCID logo http://orcid.org/0000-0002-2776-062X Donald C. Sheppard * E-mail: howell@sickkids.ca (PLH); donald.sheppard@mcgill.ca (DCS) Affiliations Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada, Infectious Diseases in Global Health Program, McGill University Health Centre, Montreal, Quebec, Canada, Department of Medicine, McGill University, Montreal, Quebec, Canada ORCID logo http://orcid.org/0000-0001-8877-880X Introduction The production of biofilms is a common strategy used by many microorganisms during infection. AMP, antimicrobial peptide; CW, cell wall; IM, inner membrane; M, membrane; NET, neutrophil extracellular trap; OM, outer membrane; PAMP, pathogen-associated molecular pattern; PG, peptidoglycan; PRR, pattern recognition receptor. https://doi.org/10.1371/journal.ppat.1007411.g001 Partially deacetylated, cationic hexosamine polymers are common in biofilm forming microorganisms A wide range of medically important microbial species produce and secrete hexosamine-rich exopolysaccharides into their self-produced extracellular biofilm matrices (Table 1). The gram-negative opportunistic pathogen Pseudomonas aeruginosa produces several biofilm-associated exopolysaccharides, including the linear heteropolymer Pel, composed of GlcNAc and N-acetyl galactosamine (GalNAc), whereas the gram-positive organism Listeria monocytogenes produces a β-1,4-linked N-acetylmannosamine polysaccharide decorated with terminal α-1,6-linked galactose (Gal) residues [5,6]. Mutants of E. coli, L. monocytogenes, P. aeruginosa, Y. pestis, Staphylococcus spp., Bordetella bronchiseptica, and A. fumigatus deficient in their respective exopolysaccharide deacetylase were found to lack detectable cell-wall–associated polysaccharide [6,8,10–12,15].

Many fungi that cause invasive disease invade host epithelial cells during mucosal and respiratory infection, and subsequently invade endothelial cells during hematogenous infection. Most fungi invade these normally non-phagocytic host cells by inducing their own uptake. Candida albicans hyphae interact with endothelial cells in vitro by binding to N-cadherin on the endothelial cell surface. This binding induces rearrangement of endothelial cell microfilaments, which results in the endocytosis of the organism. The capsule of Cryptococcus neoformans is composed of glucuronoxylomannan, which binds specifically to brain endothelial cells, and appears to mediate both adherence and induction of endocytosis. The mechanisms by which other fungal pathogens induce their own uptake are largely unknown. Some angioinvasive fungi, such as Aspergillus species and the Zygomycetes, invade endothelial cells from the abluminal surface during the initiation of invasive disease, and subsequently invade the luminal surface of endothelial cells during hematogenous dissemination. Invasion of normally non-phagocytic host cells has different consequences, depending on the type of invading fungus. Aspergillus fumigatus blocks apoptosis of pulmonary epithelial cells, whereas Paracoccidioides brasiliensis induces apoptosis of epithelial cells. This review summarizes the mechanisms by which diverse fungal pathogens invade normally non-phagocytic host cells and discusses gaps in our knowledge that provide opportunities for future research.

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus . Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes. The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus . Here, the authors study an A. fumigatus enzyme that deacetylates GAG in a metal-dependent manner and constitutes a founding member of a new carbohydrate esterase family.

Poly-β(1,6)-N-acetyl-D-glucosamine (PNAG) is a major biofilm component of many pathogenic bacteria. The production, modification, and export of PNAG in Escherichia coli and Bordetella species require the protein products encoded by the pgaABCD operon. PgaB is a two-domain periplasmic protein that contains an N-terminal deacetylase domain and a C-terminal PNAG binding domain that is critical for export. However, the exact function of the PgaB C-terminal domain remains unclear. Herein, we show that the C-terminal domains of Bordetella bronchiseptica PgaB (PgaBBb) and E. coli PgaB (PgaBEc) function as glycoside hydrolases. These enzymes hydrolyze purified deacetylated PNAG (dPNAG) from Staphylococcus aureus, disrupt PNAG-dependent biofilms formed by Bordetella pertussis, Staphylococcus carnosus, Staphylococcus epidermidis, and E. coli, and potentiate bacterial killing by gentamicin. Furthermore, we found that PgaBBb was only able to hydrolyze PNAG produced in situ by the E. coli PgaCD synthase complex when an active deacetylase domain was present. Mass spectrometry analysis of the PgaB-hydrolyzed dPNAG substrate showed a GlcN-GlcNAc-GlcNAc motif at the new reducing end of detected fragments. Our 1.76 Å structure of the C-terminal domain of PgaBBb reveals a central cavity within an elongated surface groove that appears ideally suited to recognize the GlcN-GlcNAc-GlcNAc motif. The structure, in conjunction with molecular modeling and site directed mutagenesis led to the identification of the dPNAG binding subsites and D474 as the probable catalytic acid. This work expands the role of PgaB within the PNAG biosynthesis machinery, defines a new glycoside hydrolase family GH153, and identifies PgaB as a possible therapeutic agent for treating PNAG-dependent biofilm infections.

Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall β-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.