Unsaturated fatty acid perturbation combats emerging triazole antifungal resistance in the human fungal pathogen Aspergillus fumigatus.

Contemporary antifungal therapies utilized to treat filamentous fungal infections are inhibited by intrinsic and emerging drug resistance. Consequently, there is an urgent need to develop novel antifungal compounds that are effective against drug-resistant filamentous fungi. Here, we utilized an Aspergillus fumigatus cell-based high-throughput screen to identify small molecules with antifungal activity that also potentiated triazole activity. The screen identified 16 hits with promising activity against A. fumigatus. A nonspirocyclic piperidine, herein named MBX-7591, exhibited synergy with triazole antifungal drugs and activity against pan-azole-resistant A. fumigatus isolates. MBX-7591 has additional potent activity against Rhizopus species and CO2-dependent activity against Cryptococcus neoformans. Chemical, genetic, and biochemical mode of action analyses revealed that MBX-7591 increases cell membrane saturation by decreasing oleic acid content. MBX-7591 has low toxicity in vivo and shows good efficacy in decreasing fungal burden in a murine model of invasive pulmonary aspergillosis. Taken together, our results suggest MBX-7591 is a promising hit with a novel mode of action for further antifungal drug development to combat the rising incidence of triazole-resistant filamentous fungal infections.IMPORTANCEThe incidence of infections caused by fungi continues to increase with advances in medical therapies. Unfortunately, antifungal drug development has not kept pace with the incidence and importance of fungal infections, with only three major classes of antifungal drugs currently available for use in the clinic. Filamentous fungi, also called molds, are particularly recalcitrant to contemporary antifungal therapies. Here, a recently developed Aspergillus fumigatus cell reporter strain was utilized to conduct a high-throughput screen to identify small molecules with antifungal activity. An emphasis was placed on small molecules that potentiated the activity of contemporary triazole antifungals and led to the discovery of MBX-7591. MBX-7591 potentiates triazole activity against drug-resistant molds such as A. fumigatus and has activity against Mucorales fungi. MBX-7591's mode of action involves inhibiting the conversion of saturated to unsaturated fatty acids, thereby impacting fungal membrane integrity. MBX-7591 is a novel small molecule with antifungal activity poised for lead development.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Intestinal Bacteroides modulates inflammation, systemic cytokines, and microbial ecology via propionate in a mouse model of cystic fibrosis.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1ß-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity.

IMPORTANCE: Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk for IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with chronic granulomatous disease (CGD). However, treatments for Aspergillus infections remain limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization classified A. fumigatus as a critical priority fungal pathogen. Our cell death research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

IMPORTANCE: PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Recent Advances in Understanding the Human Fungal Pathogen Hypoxia Response in Disease Progression.

Fungal-mediated disease progression and antifungal drug efficacy are significantly impacted by the dynamic infection microenvironment. At the site of infection, oxygen often becomes limiting and induces a hypoxia response in both the fungal pathogen and host cells. The fungal hypoxia response impacts several important aspects of fungal biology that contribute to pathogenesis, virulence, antifungal drug susceptibility, and ultimately infection outcomes. In this review, we summarize recent advances in understanding the molecular mechanisms of the hypoxia response in the most common human fungal pathogens, discuss potential therapeutic opportunities, and highlight important areas for future research.

Aspergillus fumigatus cytochrome c impacts conidial survival during sterilizing immunity.

Invasive pulmonary aspergillosis (IPA) is a life-threatening infection caused by species in the ubiquitous fungal genus Aspergillus . While leukocyte-generated reactive oxygen species (ROS) are critical for the clearance of fungal conidia from the lung and resistance to IPA, the processes that govern ROS-dependent fungal cell death remain poorly defined. Using a flow cytometric approach that monitors two independent cell death markers, an endogenous histone H2A:mRFP nuclear integrity reporter and Sytox Blue cell impermeable (live/dead) stain, we observed that loss of A. fumigatus cytochrome c ( cycA ) results in reduced susceptibility to cell death from hydrogen peroxide (H 2 O 2 ) treatment. Consistent with these observations in vitro , loss of cycA confers resistance to both NADPH-oxidase -dependent and -independent killing by host leukocytes. Fungal ROS resistance is partly mediated in part by Bir1, a homolog to survivin in humans, as Bir1 overexpression results in decreased ROS-induced conidial cell death and reduced killing by innate immune cells in vivo . We further report that overexpression of the Bir1 N-terminal BIR domain in A. fumigatus conidia results in altered expression of metabolic genes that functionally converge on mitochondrial function and cytochrome c ( cycA ) activity. Together, these studies demonstrate that cycA in A. fumigatus contributes to cell death responses that are induced by exogenous H 2 O 2 and by host leukocytes. Importance: Aspergillus fumigatus can cause a life-threatening infection known as invasive pulmonary aspergillosis (IPA), which is marked by fungus-attributable mortality rates of 20%-30%. Individuals at risk of IPA harbor genetic mutations or incur pharmacologic defects that impair myeloid cell numbers and/or function, exemplified by bone marrow transplant recipients, patients that receive corticosteroid therapy, or patients with Chronic Granulomatous Disease (CGD). However, treatments for Aspergillus infections remains limited, and resistance to the few existing drug classes is emerging. Recently, the World Health Organization (WHO) classified A. fumigatus as a critical priority fungal pathogen. Our research identifies an important aspect of fungal biology that impacts susceptibility to leukocyte killing. Furthering our understanding of mechanisms that mediate the outcome of fungal-leukocyte interactions will increase our understanding of both the underlying fungal biology governing cell death and innate immune evasion strategies utilized during mammalian infection pathogenesis. Consequently, our studies are a critical step toward leveraging these mechanisms for novel therapeutic advances.

Luciferase-Based High-Throughput Screen with Aspergillus fumigatus to Identify Antifungal Small Molecules.

Only three classes of contemporary antifungal drugs are routinely utilized in the clinic against filamentous fungal pathogens such as Aspergillus fumigatus. High-throughput phenotypic screens to identify small molecules with activity against filamentous fungi remain challenging due to the hyphal, biofilm-like growth morphology of these important organisms. In this chapter, we describe a protocol for utilizing a bioluminescent A. fumigatus strain for identifying small molecules that potentiate the activity of the triazole antifungal drug fluconazole. The assay holds great promise for identifying small molecules with activity against filamentous fungal pathogens.

The pan-genome of Aspergillus fumigatus provides a high-resolution view of its population structure revealing high levels of lineage-specific diversity driven by recombination.

Aspergillus fumigatus is a deadly agent of human fungal disease where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics with the potential to address the full genetic repertoire of a species. Here, we evaluate 260 genome sequences of A. fumigatus including 62 newly sequenced strains, using a combination of population genomics, phylogenomics, and pan-genomics. Our results offer a high-resolution assessment of population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes. Although A. fumigatus disperses primarily via asexual conidia, we identified extraordinarily high levels of recombination with the lowest linkage disequilibrium decay value reported for any fungal species to date. We provide evidence for 3 primary populations of A. fumigatus, with recombination occurring only rarely between populations and often within them. These 3 populations are structured by both gene variation and distinct patterns of gene presence-absence with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism suggesting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Altogether, the pan-genome of A. fumigatus represents one of the largest fungal pan-genomes reported to date including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome-based approach for evaluating intraspecific variation and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Postinfluenza Environment Reduces Aspergillus fumigatus Conidium Clearance and Facilitates Invasive Aspergillosis In Vivo.

Aspergillus fumigatus is a human fungal pathogen that is most often avirulent in immunecompetent individuals because the innate immune system is efficient at eliminating fungal conidia. However, recent clinical observations have shown that severe influenza A virus (IAV) infection can lead to secondary A. fumigatus infections with high mortality. Little is currently known about how IAV infection alters the innate antifungal immune response. Here, we established a murine model of IAV-induced A. fumigatus (IAV-Af) superinfection by inoculating mice with IAV followed 6 days later by A. fumigatus conidia challenge. We observed increased mortality in the IAV-Af-superinfected mice compared to mice challenged with either IAV or A. fumigatus alone. A. fumigatus conidia were able to germinate and establish a biofilm in the lungs of the IAV-Af superinfection group, which was not seen following fungal challenge alone. While we did not observe any differences in inflammatory cell recruitment in the IAV-Af superinfection group compared to single-infection controls, we observed defects in Aspergillus conidial uptake and killing by both neutrophils and monocytes after IAV infection. pHrodo Green zymosan bioparticle (pHrodo-zymosan) and CM-H2DCFDA [5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate] staining, indicators of phagolysosome maturation and reactive oxygen species (ROS) production, respectively, revealed that the fungal killing defect was due in part to reduced phagolysosome maturation. Collectively, our data demonstrate that the ability of neutrophils and monocytes to kill and clear Aspergillus conidia is strongly reduced in the pulmonary environment of an IAV-infected lung, which leads to invasive pulmonary aspergillosis and increased overall mortality in our mouse model, recapitulating what is observed clinically in humans. IMPORTANCE Influenza A virus (IAV) is a common respiratory virus that causes seasonal illness in humans, but can cause pandemics and severe infection in certain patients. Since the emergence of the 2009 H1N1 pandemic strains, there has been an increase in clinical reports of IAV-infected patients in the intensive care unit (ICU) developing secondary pulmonary aspergillosis. These cases of flu-Aspergillus superinfections are associated with worse clinical outcomes than secondary bacterial infections in the setting of IAV. To date, we have a limited understanding of the cause(s) of secondary fungal infections in immunocompetent hosts. IAV-induced modulation of cytokine production and innate immune cellular function generates a unique immune environment in the lung, which could make the host vulnerable to a secondary fungal infection. Our work shows that defects in phagolysosome maturation in neutrophils and monocytes after IAV infection impair the ability of these cells to kill A. fumigatus, thus leading to increased fungal germination and growth and subsequent invasive aspergillosis. Our work lays a foundation for future mechanistic studies examining the exact immune modulatory events occurring in the respiratory tract after viral infection leading to secondary fungal infections.

A Fungal Sterylglucosidase at the Intersection of Virulence, Host Immunity, and Therapeutic Development.

Human fungal infections (mycoses) cause significant morbidity and mortality in high-risk populations. Contemporary antifungal therapies rely heavily on three classes of antifungal drugs, and to date, no fungal vaccine is in clinical use for invasive mycosis. A major gap in knowledge related to fungal vaccine development is identifying lasting mechanisms of protective immunity in immunocompromised individuals. Recent studies in Cryptococcus neoformans and now Aspergillus fumigatus have identified a fungal sterylglucosidase essential for pathogenesis and virulence in murine models of mycoses. Fungal strains deficient in this sterylglucosidase can surprisingly also induce substantial immune-mediated protection against subsequent challenge with wild-type strains in multiple immunocompromised murine models of mycoses. Here, I discuss the implications and future directions of these exciting and impactful results.

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.

CF-Seq, an accessible web application for rapid re-analysis of cystic fibrosis pathogen RNA sequencing studies.

Researchers studying cystic fibrosis (CF) pathogens have produced numerous RNA-seq datasets which are available in the gene expression omnibus (GEO). Although these studies are publicly available, substantial computational expertise and manual effort are required to compare similar studies, visualize gene expression patterns within studies, and use published data to generate new experimental hypotheses. Furthermore, it is difficult to filter available studies by domain-relevant attributes such as strain, treatment, or media, or for a researcher to assess how a specific gene responds to various experimental conditions across studies. To reduce these barriers to data re-analysis, we have developed an R Shiny application called CF-Seq, which works with a compendium of 128 studies and 1,322 individual samples from 13 clinically relevant CF pathogens. The application allows users to filter studies by experimental factors and to view complex differential gene expression analyses at the click of a button. Here we present a series of use cases that demonstrate the application is a useful and efficient tool for new hypothesis generation. (CF-Seq: http://scangeo.dartmouth.edu/CFSeq/ ).

Avian-associated Aspergillus fumigatus displays broad phylogenetic distribution, no evidence for host specificity, and multiple genotypes within epizootic events.

Birds are highly susceptible to aspergillosis, which can manifest as a primary infection in both domestic and wild birds. Aspergillosis in wild birds causes mortalities ranging in scale from single animals to large-scale epizootic events. However, pathogenicity factors associated with aspergillosis in wild birds have not been examined. Specifically, it is unknown whether wild bird-infecting strains are host-adapted (i.e. phylogenetically related). Similarly, it is unknown whether epizootics are driven by contact with clonal strains that possess unique pathogenic or virulence properties, or by distinct and equally pathogenic strains. Here, we use a diverse collection of Aspergillus fumigatus isolates taken from aspergillosis-associated avian carcasses, representing 24 bird species from a wide geographic range, and representing individual bird mortalities as well as epizootic events. These isolates were sequenced and analyzed along with 130 phylogenetically diverse human clinical isolates to investigate the genetic diversity and phylogenetic placement of avian-associated A. fumigatus, the geographic and host distribution of avian isolates, evidence for clonal outbreaks among wild birds, and the frequency of azole resistance in avian isolates. We found that avian isolates were phylogenetically diverse, with no clear distinction from human clinical isolates, and no sign of host or geographic specificity. Avian isolates from the same epizootic events were diverse and phylogenetically distant, suggesting that avian aspergillosis is not contagious among wild birds and that outbreaks are likely driven by environmental spore loads or host comorbidities. Finally, all avian isolates were susceptible to Voriconazole and none contained the canonical azole resistance gene variants.

An Alanine Aminotransferase Is Required for Biofilm-Specific Resistance of Aspergillus fumigatus to Echinocandin Treatment.

Alanine metabolism has been suggested as an adaptation strategy to oxygen limitation in organisms ranging from plants to mammals. Within the pulmonary infection microenvironment, Aspergillus fumigatus forms biofilms with steep oxygen gradients defined by regions of oxygen limitation. An alanine aminotransferase, AlaA, was observed to function in alanine catabolism and is required for several aspects of A. fumigatus biofilm physiology. Loss of alaA, or its catalytic activity, results in decreased adherence of biofilms through a defect in the maturation of the extracellular matrix polysaccharide galactosaminogalactan (GAG). Additionally, exposure of cell wall polysaccharides is also impacted by loss of alaA, and loss of AlaA catalytic activity confers increased biofilm susceptibility to echinocandin treatment, which is correlated with enhanced fungicidal activity. The increase in echinocandin susceptibility is specific to biofilms, and chemical inhibition of alaA by the alanine aminotransferase inhibitor ß-chloro-l-alanine is sufficient to sensitize A. fumigatus biofilms to echinocandin treatment. Finally, loss of alaA increases susceptibility of A. fumigatus to in vivo echinocandin treatment in a murine model of invasive pulmonary aspergillosis. Our results provide insight into the interplay of metabolism, biofilm formation, and antifungal drug resistance in A. fumigatus and describe a mechanism of increasing susceptibility of A. fumigatus biofilms to the echinocandin class of antifungal drugs. IMPORTANCE Aspergillus fumigatus is a ubiquitous filamentous fungus that causes an array of diseases depending on the immune status of an individual, collectively termed aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis (IPA) or chronic pulmonary aspergillosis (CPA) is limited and too often ineffective. This is in part due to A. fumigatus biofilm formation within the infection environment and the resulting emergent properties, particularly increased antifungal resistance. Thus, insights into biofilm formation and mechanisms driving increased antifungal drug resistance are critical for improving existing therapeutic strategies and development of novel antifungals. In this work, we describe an unexpected observation where alanine metabolism, via the alanine aminotransferase AlaA, is required for several aspects of A. fumigatus biofilm physiology, including resistance of A. fumigatus biofilms to the echinocandin class of antifungal drugs. Importantly, we observed that chemical inhibition of alanine aminotransferases is sufficient to increase echinocandin susceptibility and that loss of alaA increases susceptibility to echinocandin treatment in a murine model of IPA. AlaA is the first gene discovered in A. fumigatus that confers resistance to an antifungal drug specifically in a biofilm context.

Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Aspergillus fumigatus In-Host HOG Pathway Mutation for Cystic Fibrosis Lung Microenvironment Persistence.

The prevalence of Aspergillus fumigatus colonization in individuals with cystic fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole-genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low-oxygen culture environment, and conidia were more sensitive to oxidative stress-inducing conditions than those from nonpersistent isolates. Closely related persistent isolates harbored a unique allele of the high-osmolarity glycerol (HOG) pathway mitogen-activated protein kinase kinase, Pbs2 (pbs2C2). Data suggest this novel pbs2C2 allele arose in vivo and is necessary for the fungal response to osmotic stress in a low-oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through pbs2C2 comes at the cost of decreased conidial stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent Aspergillus CF isolates. IMPORTANCE Aspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates from a patient with CF identified a persistent lineage that harbors a unique allele of the Pbs2 mitogen-activated protein kinase kinase (MAPKK) necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased fitness under CF lung-like conditions at a cost of reduced in vitro growth under standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Aspergillus fumigatus biofilms: Toward understanding how growth as a multicellular network increases antifungal resistance and disease progression.

Aspergillus fumigatus is a saprophytic, filamentous fungus found in soils and compost and the causative agent of several pulmonary diseases in humans, birds, and other mammals. A. fumigatus and other filamentous fungi grow as networks of filamentous hyphae that have characteristics of a classic microbial biofilm. These characteristics include production of an extracellular matrix (ECM), surface adhesion, multicellularity, and increased antimicrobial drug resistance. A. fumigatus biofilm growth occurs in vivo at sites of infection, highlighting the importance of defining mechanisms underlying biofilm development and associated emergent properties. We propose that there are 3 distinct phases in the development of A. fumigatus biofilms: biofilm initiation, immature biofilm, and mature biofilm. These stages are defined both temporally and by unique genetic and structural changes over the course of development. Here, we review known mechanisms within each of these stages that contribute to biofilm structure, ECM production, and increased resistance to contemporary antifungal drugs. We highlight gaps in our understanding of biofilm development and function that when addressed are expected to aid in the development of novel antifungal therapies capable of killing filamentous fungal biofilms.

A Heterogeneously Expressed Gene Family Modulates the Biofilm Architecture and Hypoxic Growth of Aspergillus fumigatus.

The genus Aspergillus encompasses human pathogens such as Aspergillus fumigatus and industrial powerhouses such as Aspergillus niger In both cases, Aspergillus biofilms have consequences for infection outcomes and yields of economically important products. However, the molecular components influencing filamentous fungal biofilm development, structure, and function remain ill defined. Macroscopic colony morphology is an indicator of underlying biofilm architecture and fungal physiology. A hypoxia-locked colony morphotype of A. fumigatus has abundant colony furrows that coincide with a reduction in vertically oriented hyphae within biofilms and increased low oxygen growth and virulence. Investigation of this morphotype has led to the identification of the causative gene, biofilm architecture factor A (bafA), a small cryptic open reading frame within a subtelomeric gene cluster. BafA is sufficient to induce the hypoxia-locked colony morphology and biofilm architecture in A. fumigatus Analysis across a large population of A. fumigatus isolates identified a larger family of baf genes, all of which have the capacity to modulate hyphal architecture, biofilm development, and hypoxic growth. Furthermore, introduction of A. fumigatusbafA into A. niger is sufficient to generate the hypoxia-locked colony morphology, biofilm architecture, and increased hypoxic growth. Together, these data indicate the potential broad impacts of this previously uncharacterized family of small genes to modulate biofilm architecture and function in clinical and industrial settings.IMPORTANCE The manipulation of microbial biofilms in industrial and clinical applications remains a difficult task. The problem is particularly acute with regard to filamentous fungal biofilms for which molecular mechanisms of biofilm formation, maintenance, and function are only just being elucidated. Here, we describe a family of small genes heterogeneously expressed across Aspergillus fumigatus strains that are capable of modifying colony biofilm morphology and microscopic hyphal architecture. Specifically, these genes are implicated in the formation of a hypoxia-locked colony morphotype that is associated with increased virulence of A. fumigatus Synthetic introduction of these gene family members, here referred to as biofilm architecture factors, in both A. fumigatus and A. niger additionally modulates low oxygen growth and surface adherence. Thus, these genes are candidates for genetic manipulation of biofilm development in aspergilli.