Development of a murine model to study the cerebral pathogenesis of Aspergillus fumigatus.

IMPORTANCE: Molds are environmental fungi that can cause disease in immunocompromised individuals. The most common pathogenic mold is Aspergillus fumigatus, which is typically inhaled into the lungs and causes invasive pulmonary disease. In a subset of these patients, this infection can spread from the lungs to other organs including the brain, resulting in cerebral aspergillosis. How A. fumigatus causes brain disease is not well understood and these infections are associated with extremely high mortality rates. Thus, we developed an animal model to study the pathogenesis of cerebral aspergillosis to better understand this disease and develop better treatments for these life-threatening infections.

The anti-cancer efficacy of a novel phenothiazine derivative is independent of dopamine and serotonin receptor inhibition.

Introduction: An attractive, yet unrealized, goal in cancer therapy is repurposing psychiatric drugs that can readily penetrate the blood-brain barrier for the treatment of primary brain tumors and brain metastases. Phenothiazines (PTZs) have demonstrated anti-cancer properties through a variety of mechanisms. However, it remains unclear whether these effects are entirely separate from their activity as dopamine and serotonin receptor (DR/5-HTR) antagonists. Methods: In this study, we evaluated the anti-cancer efficacy of a novel PTZ analog, CWHM-974, that was shown to be 100-1000-fold less potent against DR/5-HTR than its analog fluphenazine (FLU). Results: CWHM-974 was more potent than FLU against a panel of cancer cell lines, thus clearly demonstrating that its anti-cancer effects were independent of DR/5-HTR signaling. Our results further suggested that calmodulin (CaM) binding may be necessary, but not sufficient, to explain the anti-cancer effects of CWHM-974. While both FLU and CWHM-974 induced apoptosis, they induced distinct effects on the cell cycle (G0/G1 and mitotic arrest respectively) suggesting that they may have differential effects on CaM-binding proteins involved in cell cycle regulation. Discussion: Altogether, our findings indicated that the anti-cancer efficacy of the CWHM-974 is separable from DR/5-HTR antagonism. Thus, reducing the toxicity associated with phenothiazines related to DR/5-HTR antagonism may improve the potential to repurpose this class of drugs to treat brain tumors and/or brain metastasis.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP-Binding Cassette (ABC) and Major Facilitator Superfamily proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased MDR transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of Candida drug resistance (CDR) transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata, despite inducing CDR1 expression to high levels. Overall, CWHM-974 is one of the few examples of a molecule in which relatively small structural modifications significantly reduced susceptibility to multidrug transporter-mediated resistance.

CWHM-974 is a fluphenazine derivative with improved antifungal activity against Candida albicans due to reduced susceptibility to multidrug transporter-mediated resistance mechanisms.

Multidrug resistance (MDR) transporters such as ATP Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) proteins are important mediators of antifungal drug resistance, particularly with respect to azole class drugs. Consequently, identifying molecules that are not susceptible to this mechanism of resistance is an important goal for new antifungal drug discovery. As part of a project to optimize the antifungal activity of clinically used phenothiazines, we synthesized a fluphenazine derivative (CWHM-974) with 8-fold higher activity against Candida spp. compared to the fluphenazine and with activity against Candida spp. with reduced fluconazole susceptibility due to increased multidrug resistance transporters. Here, we show that the improved C. albicans activity is because fluphenazine induces its own resistance by triggering expression of CDR transporters while CWHM-974 induces expression but does not appear to be a substrate for the transporters or is insensitive to their effects through other mechanisms. We also found that fluphenazine and CWHM-974 are antagonistic with fluconazole in C. albicans but not in C. glabrata , despite inducing CDR1 expression to high levels. Overall, CWHM-974 represents a unique example of a medicinal chemistry-based conversion of chemical scaffold from MDR-sensitive to MDR-resistant and, hence, active against fungi that have developed resistance to clinically used antifungals such as the azoles.

Novel Keto-Alkyl-Pyridinium Antifungal Molecules Active in Models of In Vivo Candida albicans Vascular Catheter Infection and Ex Vivo Candida auris Skin Colonization.

New antifungal therapies are needed for both systemic, invasive infections in addition to superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to nonsystemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2 to 16 µg/mL) against medically important yeasts and molds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus. Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce Candida albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial preclinical data suggest that molecules of this class may warrant further study and development for nonsystemic applications.

A Dual-Readout High-Throughput Screening Assay for Small Molecules Active Against Aspergillus Fumigatus.

Human fungal infections caused by molds have been on the rise in recent years. These infections have high mortality rates compared to other fungal infections, and yet treatment options are limited due to resistance to clinical antifungals and lack of broad-spectrum activity against molds. Technical challenges associated with molds have limited large-throughput screening efforts for anti-mold compounds: therefore, we adapted an assay for use with A. fumigatus to help fill the gap in robust screening platforms for these organisms. This assay measures the release of the cytosolic enzyme adenylate kinase (AK) as a measure of fungal cell lysis and can also detect inhibition of germination as a reduction in the secretion of AK during vegetative growth. The ability to detect both lysis and inhibition of germination facilitates the identification of a wide range of compounds with different mechanisms of action, creating a strong screening platform for the identification of novel, anti-mold compounds.

Novel keto-alkyl-pyridinium antifungal molecules active in models of in vivo Candida albicans vascular catheter infection and ex vivo Candida auris skin colonization.

New antifungal therapies are needed for both systemic, invasive infections as well as superficial infections of mucosal and skin surfaces as well as biofilms associated with medical devices. The resistance of biofilm and biofilm-like growth phases of fungi contributes to the poor efficacy of systemic therapies to non-systemic infections. Here, we describe the identification and characterization of a novel keto-alkyl-pyridinium scaffold with broad spectrum activity (2-16 µg/mL) against medically important yeasts and moulds, including clinical isolates resistant to azoles and/or echinocandins. Furthermore, these keto-alkyl-pyridinium agents retain substantial activity against biofilm phase yeast and have direct activity against hyphal A. fumigatus . Although their toxicity precludes use in systemic infections, we found that the keto-alkyl-pyridinium molecules reduce C. albicans fungal burden in a rat model of vascular catheter infection and reduce Candida auris colonization in a porcine ex vivo model. These initial pre-clinical data suggest that molecules of this class may warrant further study and development.

Genetic Interaction Analysis Reveals that Cryptococcus neoformans Utilizes Multiple Acetyl-CoA-Generating Pathways during Infection.

Cryptococcus neoformans is an important human fungal pathogen for which the external environment is its primary niche. Previous work has shown that two nonessential acetyl-CoA metabolism enzymes, ATP-citrate lyase (ACL1) and acetyl-CoA synthetase (ACS1), play important roles in C. neoformans infection. Here, we took a genetic interaction approach to studying the interplay between these two enzymes along with an enzyme initially called ACS2 but which we have found is an acetoacetyl-CoA synthetase; we have renamed the gene 2-ketobutyryl CoA synthetase 1 (KBC1) based on its biochemical activity and the systematic name of its substrate. ACL1 and ACS1 represent a synthetic lethal pair of genes based on our genetic interaction studies. Double mutants of KBC1 with either ACS1 or ACL1 do not have significant synthetic phenotypes in vitro, although we find that deletion of any one of these enzymes reduces fitness within macrophages. Importantly, the acs1Δ kbc1Δ double mutant has significantly reduced fitness in the CNS relative to either single mutant as well as WT (~2 log10 CFU reduction in fungal burden), indicating the important role these enzymes play during infection. The expression of both ACS1 and KBC1 is increased in vivo relative to in vitro conditions. The acs1Δ mutant is hypersusceptible to fluconazole in vivo despite its minimal in vitro phenotypes. These data not only provide insights into the in vivo mechanism of action for a new class of antifungal Acs inhibitors but also into metabolic adaptations of C. neoformans to the host environment. IMPORTANCE The adaptation of environmental fungal pathogens to the mammalian host is critical to pathogenesis. Of these adaptations, the remodeling of carbon metabolism is particularly important. Here, we generated a focused set of double mutants of nonessential genes (ACL1, ACS1, KBC1) involved in acetyl-CoA metabolism and examined their fitness in vitro and during CNS infection. From these studies, we found that all three enzymes play important roles during infection but that the role of ACS1/KBC1 was minimal in vitro. Consistent with these observations, the expression of ACS1 and KBC1 was increased in vivo relative to standard in vitro conditions. Furthermore, strains lacking both ACL1 and ACS1 were not viable. These data clearly show that C. neoformans employs multiple carbon metabolism pathways to adapt to the host environment. They also provide insights into the potential mechanism of action for anti-cryptococcal Acs inhibitors.

FKS1 Is Required for Cryptococcus neoformans Fitness In Vivo: Application of Copper-Regulated Gene Expression to Mouse Models of Cryptococcosis.

There is an urgent need for new antifungals to treat cryptococcal meningoencephalitis, a leading cause of mortality in people living with HIV/AIDS. An important aspect of antifungal drug development is the validation of targets to determine whether they are required for the survival of the organism in animal models of disease. In Cryptococcus neoformans, a copper-regulated promoter (pCTR4-2) has been used previously to modulate gene expression in vivo. The premise for these experiments is that copper concentrations differ depending on the host niche. Here, we directly test this premise and confirm that the expression of CTR4, the promoter used to regulate gene expression, is much lower in the mouse lung compared to the brain. To further explore this approach, we applied it to the gene encoding 1,3-ß-glucan synthase, FKS1. In vitro, reduced expression of FKS1 has little effect on growth but does activate the cell wall integrity stress response and increase susceptibility to caspofungin, a direct inhibitor of Fks1. These data suggest that compensatory pathways that reduce C. neoformans resistance do so through posttranscriptional effects. In vivo, however, a less pronounced reduction in FKS1 expression leads to a much more significant reduction in lung fungal burden (~1 log10 CFU), indicating that the compensatory responses to a reduction in FKS1 expression are not as effective in vivo as they are in vitro. In summary, use of copper-regulated expression of putative drug targets in vitro and in vivo can provide insights into the biological consequences of reduced activity of the target during infection. IMPORTANCE Conditional expression systems are widely used to genetically validate antifungal drug targets in mouse models of infection. Copper-regulated expression using the promoter of the CTR4 gene has been sporadically used for this purpose in C. neoformans. Here, we show that CTR4 expression is low in the lung and high in the brain, establishing the basic premise behind this approach. We applied the approach to the study of FKS1, the gene encoding the target of the echinocandin class of 1,3-ß-glucan synthase inhibitors. Our in vitro and in vivo studies indicate that C. neoformans tolerates extremely low levels of FKS1 expression. This observation provides a potential explanation for the poor activity of 1,3-ß-glucan synthase inhibitors toward C. neoformans.

Gluconate Kinase Is Required for Gluconate Assimilation and Sporulation in Cryptococcus neoformans.

Cryptococcus neoformans is an environmental yeast and an opportunistic human pathogen. The ability to cause disease depends on the ability to adapt to the human host. Previous studies implicated infectivity-related kinase 3 (IRK3, CNAG_03048) as required for establishing an infection. We genetically and biochemically characterized IRK3 as a gluconate kinase and propose the name GNK1. This metabolic enzyme utilizes gluconate to produce 6-phosphogluconate as part of the alternative oxidative phase of the pentose phosphate pathway (AOXPPP). The presence of GNK1 confirms that the AOXPPP is present and able to compensate for loss of the traditional OXPPP, providing an explanation for its nonessentiality. C. neoformans can utilize gluconate as an alternative carbon source in a GNK1-dependent manner. In our efforts to understand the role of GNK1 in host adaptation and virulence, we found that GNK1-deficient mutants have variable virulence and carbon dioxide tolerance across multiple strains, suggesting that second site mutations frequently interact with GNK1 deletion mutations. In our effort to isolate these genetic loci by backcrossing experiments, we discovered that GNK1-deficient strains are unable to sporulate. These data suggest that gluconate metabolism is critical for sporulation of C. neoformans. IMPORTANCE Cryptococcus neoformans is a fungal pathogen that contributes to nearly 180,000 deaths annually. We characterized a gene named GNK1 that appears to interact with other genetic loci involved with the ability of C. neoformans to act as a pathogen. While these interacting genetic loci remain elusive, we discovered that GNK1 plays roles in both metabolism and mating/sporulation. Further interrogation of the mechanistic role for GNK1 in sexual reproduction may uncover a larger network of genes that are important for host adaptation and virulence.

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.

Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence.

The opportunistic human fungal pathogen Cryptococcus neoformans has tremendous impact on global health, causing 181,000 deaths annually. Current treatment options are limited, and the frequent development of drug resistance exacerbates the challenge of managing invasive cryptococcal infections. In diverse fungal pathogens, the essential molecular chaperone Hsp90 governs fungal survival, drug resistance, and virulence. Therefore, targeting this chaperone has emerged as a promising approach to combat fungal infections. However, the role of Hsp90 in supporting C. neoformans pathogenesis remains largely elusive due to a lack of genetic characterization. To help dissect the functions of Hsp90 in C. neoformans, we generated a conditional expression strain in which HSP90 is under control of the copper-repressible promoter CTR4-2. Addition of copper to culture medium depleted Hsp90 transcript and protein levels in this strain, resulting in compromised fungal growth at host temperature; increased sensitivity to stressors, including the azole class of antifungals; altered C. neoformans morphology; and impaired melanin production. Finally, leveraging the fact that copper concentrations vary widely in different mouse tissues, we demonstrated attenuated virulence for the CTR4-2p-HSP90 mutant specifically in an inhalation model of Cryptococcus infection. During invasion and establishment of infection in this mouse model, the pathogen is exposed to the relatively high copper concentrations found in the lung as compared to blood. Overall, this work generates a tractable genetic system to study the role of Hsp90 in supporting the pathogenicity of C. neoformans and provides proof-of-principle that targeting Hsp90 holds great promise as a strategy to control cryptococcal infection.

A Unique Dual-Readout High-Throughput Screening Assay To Identify Antifungal Compounds with Aspergillus fumigatus.

Treatment of invasive mold infections is limited by the lack of adequate drug options that are effective against these fatal infections. High-throughput screening of molds using traditional antifungal assays of growth is problematic and has greatly limited our ability to identify new mold-active agents. Here, we present a high-throughput screening platform for use with Aspergillus fumigatus, the most common causative agent of invasive mold infections, for the discovery of novel mold-active antifungals. This assay detects cell lysis through the release of the cytosolic enzyme adenylate kinase and, thus, is not dependent on changes in biomass or metabolism to detect antifungal activity. The ability to specifically detect cell lysis is a unique aspect of this assay that allows identification of molecules that disrupt fungal cell integrity, such as cell wall-active molecules. We also found that germinating A. fumigatus conidia release low levels of adenylate kinase and that a reduction in this background allowed us to identify molecules that inhibit conidial germination, expanding the potential for discovery of novel antifungal compounds. Here, we describe the validation of this assay and proof-of-concept pilot screens that identified a novel antifungal compound, PIK-75, that disrupts cell wall integrity. This screening assay provides a novel platform for high-throughput screens with A. fumigatus for the identification of anti-mold drugs. IMPORTANCE Fungal infections caused by molds have the highest mortality rates of human fungal infections. These devastating infections are hard to treat and available antifungal drugs are often not effective. Therefore, the identification of new antifungal drugs with mold activity is critical. Drug screening with molds is challenging and there are limited assays available to identify new antifungal compounds directly with these organisms. Here, we present an assay suitable for use for high-throughput screening with a common mold pathogen. This assay has exciting future potential for the identification of new drugs to treat these fatal infections.

Enantioselective Synthesis of (+)-Hippolide J and Reevaluation of Antifungal Activity.

A synthesis of the reported antifungal agent (+)-hippolide J is presented. The rapid assembly of the natural product was enabled through implementation of an enantioselective isomerization/[2 + 2]-cycloaddition sequence. Due to the simplicity of the route, >100 mg of the natural product were prepared in a single pass. Anitfungal assays of hippolide J, however, confirmed that it showed no activity against several fungal strains, contrary to the isolation report.

Antifungal drug screening: thinking outside the box to identify novel antifungal scaffolds.

Invasive fungal infections are responsible for a significant disease burden worldwide. Drugs to treat these infections are limited to only four unique classes, and despite these available treatments, mortality rates remain unacceptably high. In this review, we will discuss antifungal drug screening and how the approach to identifying novel compounds needs move away from traditional growth-based assays in order to meet the demand for new drugs. We highlight specific examples of creative screening strategies that increase the likelihood of identifying compounds with desired activities and provide perspective to inspire development of novel screens for the identification of first-in-class antifungals.

Benzothiourea Derivatives Target the Secretory Pathway of the Human Fungal Pathogen Cryptococcus neoformans.

Cryptococcus neoformans is one of the most important human fungal pathogens and causes life-threatening meningoencephalitis in immunocompromised patients. The current gold standard therapy for C. neoformans meningoencephalitis is based on medications that are over 50 years old and is not readily available in regions with high disease burden. Here, we report the mycologic, mechanistic, and pharmacologic characterization of a set of benzothioureas with highly selective fungicidal activity against C. neoformans. In addition, to direct antifungal activity, benzothioureas inhibit C. neoformans virulence traits. On the basis of a set of phenotypic, biochemical, and biophysical assays, the benzothioureas (BTUs) inhibit the late secretory pathway (post-Golgi), possibly through a direct interaction with Sav1, an orthologue of the Sec4-class small GTPase. Importantly, pharmacological characterization of the BTUs indicates it readily penetrates the blood-brain barrier. Together, our data support the further development of this scaffold as an antifungal agent with a novel mechanism of action against C. neoformans.

Host Carbon Dioxide Concentration Is an Independent Stress for Cryptococcus neoformans That Affects Virulence and Antifungal Susceptibility.

The ability of Cryptococcus neoformans to cause disease in humans varies significantly among strains with highly related genotypes. In general, environmental isolates of pathogenic species such as Cryptococcus neoformans var. grubii have reduced virulence relative to clinical isolates, despite having no differences in the expression of the canonical virulence traits (high-temperature growth, melanization, and capsule formation). In this observation, we report that environmental isolates of C. neoformans tolerate host CO2 concentrations poorly compared to clinical isolates and that CO2 tolerance correlates well with the ability of the isolates to cause disease in mammals. Initial experiments also suggest that CO2 tolerance is particularly important for dissemination of C. neoformans from the lung to the brain. Furthermore, CO2 concentrations affect the susceptibility of both clinical and environmental C. neoformans isolates to the azole class of antifungal drugs, suggesting that antifungal testing in the presence of CO2 may improve the correlation between in vitro azole activity and patient outcome.IMPORTANCE A number of studies comparing either patient outcomes or model system virulence across large collections of Cryptococcus isolates have found significant heterogeneity in virulence even among strains with highly related genotypes. Because this heterogeneity cannot be explained by variations in the three well-characterized virulence traits (growth at host body temperature, melanization, and polysaccharide capsule formation), it has been widely proposed that additional C. neoformans virulence traits must exist. The natural niche of C. neoformans is in the environment, where the carbon dioxide concentration is very low (∼0.04%); in contrast, mammalian host tissue carbon dioxide concentrations are 125-fold higher (5%). We have found that the ability to grow in the presence of 5% carbon dioxide distinguishes low-virulence strains from high-virulence strains, even those with a similar genotype. Our findings suggest that carbon dioxide tolerance is a previously unrecognized virulence trait for C. neoformans.

Characterizing the Pathogenic, Genomic, and Chemical Traits of Aspergillus fischeri, a Close Relative of the Major Human Fungal Pathogen Aspergillus fumigatus.

Aspergillus fischeri is closely related to Aspergillus fumigatus, the major cause of invasive mold infections. Even though A. fischeri is commonly found in diverse environments, including hospitals, it rarely causes invasive disease. Why A. fischeri causes less human disease than A. fumigatus is unclear. A comparison of A. fischeri and A. fumigatus for pathogenic, genomic, and secondary metabolic traits revealed multiple differences in pathogenesis-related phenotypes. We observed that A. fischeri NRRL 181 is less virulent than A. fumigatus strain CEA10 in multiple animal models of disease, grows slower in low-oxygen environments, and is more sensitive to oxidative stress. Strikingly, the observed differences for some traits are of the same order of magnitude as those previously reported between A. fumigatus strains. In contrast, similar to what has previously been reported, the two species exhibit high genomic similarity; ∼90% of the A. fumigatus proteome is conserved in A. fischeri, including 48/49 genes known to be involved in A. fumigatus virulence. However, only 10/33 A. fumigatus biosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved in A. fischeri and only 13/48 A. fischeri BGCs are conserved in A. fumigatus Detailed chemical characterization of A. fischeri cultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Additionally, an A. fischeri deletion mutant of laeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the nonpathogenic A. fischeri possesses many of the genes important for A. fumigatus pathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.IMPORTANCEAspergillus fumigatus is the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide. A. fischeri is a close relative of A. fumigatus but is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of pathogenesis-relevant biological and chemical characteristics. We found that disease progression in multiple A. fischeri mouse models was slower and caused less mortality than A. fumigatus Remarkably, the observed differences between A. fischeri and A. fumigatus strains examined here closely resembled those previously described for two commonly studied A. fumigatus strains, AF293 and CEA10. A. fischeri and A. fumigatus exhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of chemicals that induce the production of reactive oxygen species. We also found that the vast majority of A. fumigatus genes known to be involved in virulence are conserved in A. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we report here are the first step toward understanding the evolutionary origin of a major fungal pathogen.

Response to Comment on "Sterilizing immunity in the lung relies on targeting fungal apoptosis-like programmed cell death".

Aouacheria et al question the interpretation of contemporary assays to monitor programmed cell death with apoptosis-like features (A-PCD) in Aspergillus fumigatus Although our study focuses on fungal A-PCD for host immune surveillance and infectious outcomes, the experimental approach incorporates multiple independent A-PCD markers and genetic manipulations based on fungal rather than mammalian orthologs to circumvent the limitations associated with any single approach.