Expression of a fungal-specific sub-telomeric gene, hrmA , in Aspergillus fumigatus is important for a colony biofilm morphology termed H-MORPH, increased hypoxic fitness, and virulence in a murine model of invasive pulmonary aspergillosis (IPA). How expression of hrmA contributes to virulence and worse disease progression is ill-defined. Increased hrmA expression results in reduced attachment of the extracellular matrix (ECM) to the fungal cell wall resulting in decreased strain adherence. Fungal strains that are less adherent in vitro are typically less virulent as the ECM heteropolysaccharide galactosaminogalactan (GAG) aids in adhesion to host cells and confers protection from host responses. Here we report that the UDP-glucose 4-epimerase encoding gene required for GAG biosynthesis, uge3 , is necessary for full virulence of the H-MORPH strain, hrmA REV (AF293:: hrmA D304G ). In contrast, loss of uge3 in the reference strain AF293 did not significantly impact virulence in the tested IPA murine model. Phenotypic, transcriptomic, and metabolic analyses of uge3 loss in the respective strain backgrounds revealed a key role for Uge3 in central carbon metabolism in a strain specific context that promotes disease progression. These results complement the known role of Uge3 in GAG biosynthesis and highlight strain specific metabolic differences in pathogenic A. fumigatus strains. IMPORTANCE:Aspergillus fumigatus forms adherent biofilms that contribute to its ability to persist and cause disease. However, significant strain diversity exists with regard to the morphology of A. fumigatus biofilms. A distinct colony morphotype associated with increased disease progression and low oxygen fitness, termed H-MORPH, was recently described. An additional defining feature of the H-MORPH biofilm morphotype is reduced in vitro adherence to surfaces. While reduced fungal strain adherence is most commonly associated with reductions in virulence, H-MORPH strains exhibit increased virulence relative to the well-studied N-MORPH reference strain AF293. Here we discover that the UDP-glucose 4-epimerase, Uge3, plays an important role in H-MORPH central carbon metabolism complementary to its role in production of the extracellular matrix polysaccharide galactosaminogalactan (GAG). In H-MORPH strains, this metabolic role for Uge3 becomes central to virulence. These data highlight A. fumigatus strain specific mechanisms of fungal carbon metabolism related to biofilm matrix production and fungal virulence.

Virus-associated pulmonary aspergillosis is a life-threatening secondary infection that substantially increases morbidity and mortality in critically ill patients with respiratory virus infections. Influenza A virus (IAV) and SARS-CoV2 are known to disrupt pulmonary homeostasis, the mechanisms by which these perturbations render the host susceptibility to Aspergillus fumigatus (Af) remain incompletely understood. Here, we integrate an established murine model of influenza-associated pulmonary aspergillosis (IAPA) with single-cell RNA sequencing (scRNA-seq) to define the myeloid cell dysfunction that underlies IAPA establishment and progression. Single-cell transcriptomic profiling of pulmonary monocytes and macrophages revealed that IAV-Af coinfection drives a marked shift away from interferon-mediated antiviral and antigen presentation programs toward stress-associated and redox-regulatory transcriptional states. Pathway analyses demonstrated coordinated suppression of phagocytic and interferon signaling pathways alongside enrichment of oxidative stress and mitochondrial metabolic signatures - changes that closely recapitulate transcriptional defects previously reported in human IAPA patients. Myeloid cells from IAV-Af coinfected mice further exhibited increased oxidative phosphorylation alongside reduced glycolytic and phagocytic activity, consistent with impaired antifungal effector function. To elucidate how prior IAV infection generates a pulmonary microenvironment permissive to Af growth, we evaluated airway iron availability - a critical determinant of both fungal pathogenicity and immune regulation. IAV infection alone produced a significant elevation in bronchoalveolar iron levels accompanied by induction of iron-associated inflammatory mediators. Paradoxically, during IAV-Af coinfection, myeloid cells displayed markedly reduced expression of iron-sequestering and storage genes, revealing a fundamental disconnect between iron burden and cellular iron-handling capacity. Functionally, elevated iron accelerated Af germination and impaired macrophage-mediated fungal killing. Collectively, these findings identify IAV-induced pulmonary iron accumulation as a key driver of immunometabolic reprogramming in myeloid cells, resulting in compromised antifungal immunity and heightened susceptibility to secondary Af infection.

Aspergillus fumigatus poses a significant threat to human well-being, in part due to the increasing emergence of strains resistant to frontline antifungal therapy. In this study, we observed that the gene arvA is required for A. fumigatus morphogenesis, antifungal drug susceptibility, and cell wall homeostasis. Intriguingly, our study reveals novel morphological and growth aberrations in the absence of arvA. Loss of arvA results in hyper-swollen conidia that give rise to stunted, polarity-deficient hyphae in numerous environmental conditions, indicating a pivotal role for arvA in A. fumigatus morphogenesis. Surprisingly, despite these severe in vitro morphological and cell wall defects, arvA was not required for morbidity and mortality in immunologically distinct murine models of invasive pulmonary aspergillosis. However, the mechanisms by which the arvA mutant can cause disease differ between the two models. Growth in natural calf lung surfactant was able to somewhat normalize ∆arvA growth with the wild-type strain, suggesting lung surfactant may partially complement the severe in vitro morphological defects of arvA loss in vivo. Taken together, our observations reveal arvA as a mediator of A. fumigatus antifungal drug susceptibility and highlight the complex and ill-defined pulmonary nutrient environment's role in mediating A. fumigatus pathogenesis and disease progression. IMPORTANCE:Aspergillus fumigatus is a challenging fungal pathogen in the clinic, in part due to increasing azole drug resistance. In this study, we observed that the loss of the A. fumigatus gene arvA results in increased azole susceptibility and significant in vitro morphological changes highlighted by hyper-swollen conidia that yield stunted and polarity-deficient hyphae. Importantly, despite these severe in vitro morphological and growth abnormalities, ∆arvA surprisingly retains full pathogenicity and virulence in two immunologically distinct murine models of invasive pulmonary aspergillosis. These results challenge our understanding of the in-host environment and how it mediates fungal morphogenesis and pathogenesis. These results, consequently, not only enhance our understanding of the role of arvA in A. fumigatus morphogenesis and drug susceptibility but also further emphasize the importance of in vivo animal models in fully evaluating potential antifungal drug targets.

Aspergillus fumigatus detection in respiratory samples of people with cystic fibrosis (CF) is important for the diagnosis of Aspergillus-related lung disease. Yet, fungal culture approaches are not standardized for clinical CF respiratory samples. We analyzed CF sputum to determine culture conditions that may improve the detection of Aspergillus fumigatus. A total of 281 remotely collected sputum samples from 74 adults with CF were analyzed in two laboratories, Johns Hopkins and Dartmouth. Aspergillus fumigatus was found in 121 (74%) and 84 (71%) samples in Johns Hopkins and Dartmouth, respectively. In the Johns Hopkins laboratory, we observed a decreased odds for Aspergillus fumigatus growth when using Czapek-Dox agar (adjusted OR [aOR] 0.78, 95% CI 0.68-0.89, P < 0.01) compared to inhibitory mold agar. An incubation temperature of 30°C compared to 37°C was associated with decreased Aspergillus fumigatus positivity in 21%O2 (aOR 0.71, 95% CI 0.58-0.88, P < 0.01). We also observed 82% increased odds for Aspergillus fumigatus isolation while incubating in 37°C 1% oxygen (O2) compared to 37°C 21% O25% carbon dioxide (CO2) (aOR 1.82, 95% CI 1.41-2.36, P < 0.01). Additional work in the Dartmouth laboratory demonstrated Sabouraud agar media was associated with decreased odds for Aspergillus fumigatus positive culture compared to inhibitory mold agar (aOR 0.77, 95% CI 0.63-0.93, P < 0.01). Our data suggest that use of inhibitory mold agar and 37°C and hypoxic incubation conditions may increase the odds to detect Aspergillus fumigatus in remotely collected CF sputa. Validation studies are needed to determine if these culture conditions improve A. fumigatus detection in CF. IMPORTANCE:Aspergillus fumigatus is the most common filamentous fungus that affects the lungs of people with cystic fibrosis (CF), and the detection is critical to determine whether treatment is required. We examined how fungal culture media, sputum processing, and incubation conditions may impact the recovery of Aspergillus fumigatus in CF sputum. Our study found that semi-selective fungal culture media, such as inhibitory mold agar and incubation in 37°C and low oxygen, were associated with Aspergillus fumigatus growth in remotely collected CF sputum. Validation of our findings has the potential to impact the approach of clinical fungal culture to detect Aspergillus fumigatus in the CF population.

MDA5 is a cytosolic pattern-recognition receptor (PRR) that binds to double-stranded RNA (dsRNA) and subsequently interacts with the signaling adaptor protein MAVS to initiate the antiviral interferon (IFN) response. Our group previously demonstrated that MDA5 is essential for host resistance against the fungal pathogen, Aspergillus fumigatus. Although fungal dsRNA was sufficient to activate MDA5 signaling, the precise source of A. fumigatus dsRNA responsible for this MDA5-stimulating function remains unknown. Here, we demonstrate that the magnitude of the IFN-dependent antifungal response is A. fumigatus strain dependent. Unexpectedly, we found that A. fumigatus isolates infected with dsRNA mycoviruses triggered a more robust MAVS-dependent inflammatory response within alveolar macrophages. Furthermore, dsRNA mycovirus infection increased fungal susceptibility to antifungal killing without altering other A. fumigatus growth characteristics. Although dsRNA mycovirus infection did not alter virulence in an acute bronchopneumonia model of A. fumigatus infection, it significantly impaired virulence and improved disease parameters in a chronic model of allergic bronchopulmonary aspergillosis (ABPA). Collectively, these findings reveal a novel role for trans-kingdom interactions in driving the host antifungal IFN response and modulating virulence in chronic aspergillosis models.

Antibiotic tolerance paves the way for acquired resistance in bacterial pathogens. However, the mechanisms of tolerance and its evolutionary role in acquired resistance in pathogenic fungi, and particularly in filamentous fungi, remain elusive. Here, we identified an Inhibitor of Growth domain-containing protein (IngB) as a novel epigenetic regulator of azole tolerance in Aspergillus fumigatus. The loss of ingB promotes supra-MIC growth on agar surfaces despite susceptible MICs in standardized assays. Moreover, established ΔingB biofilms are also less susceptible to azoles in vitro. In a murine model of invasive pulmonary aspergillosis, loss of ingB results in higher pulmonary fungal levels when animals are treated with voriconazole compared to the wild-type control. Subsequent exposure of the ΔingB-tolerant strain to high azole concentrations in vitro resulted in rapid acquired resistance, most notably driven by a frameshift mutation in a putative 20S proteasome maturation protein-encoding gene, umpA, while the susceptible wild-type strain failed to acquire adaptive mutations. The data suggest that loss of IngB provides an epistatic background for the emergence of azole resistance. Our work shows that drug tolerance in a critical fungal pathogen can facilitate azole resistance emergence.IMPORTANCEWhile antimicrobial drug resistance causes adverse effects on human health, drug tolerance can also lead to insufficient pathogen clearance, resulting in infection relapse. However, the mechanisms of antifungal drug tolerance and its evolutionary role in acquired drug resistance in pathogenic fungi, particularly the molds, remain elusive. We identified IngB as a novel regulator of azole tolerance in Aspergillus fumigatus. In a murine model of invasive pulmonary aspergillosis treated with voriconazole, loss of ingB facilitated higher fungal burden levels than the wild-type control, suggesting the observed in vitro tolerance translates to the murine pulmonary environment. Importantly, loss of IngB leads to rapid azole drug resistance under azole-selective pressure in vitro and led to the discovery of a new gene associated with azole resistance, umpA. Our work identifies a novel regulator of antifungal tolerance in a critical human fungal pathogen and suggests that drug tolerance can pave the way for resistance emergence.

The complex structure of fungal biofilms generates microenvironments that impact the fitness of cells within the biofilm community. Contributions to fitness include the development of emergent properties resulting in the tolerance or resistance to external stressors, such as rapid environmental changes and, in the context of an infection, antifungal drug exposure. The biofilm developed by the filamentous fungal pathogen Aspergillus fumigatus develops zones of low oxygen, which contribute to a reduction in antifungal drug susceptibility. The genes and mechanisms involved in driving this biofilm-specific emergent property are ill-defined. In this study, we utilized a transcriptomic approach to probe the biofilm structure in comparison to drug-susceptible planktonic cultures to identify transcriptional patterns and genes unique to the A. fumigatus biofilm. Importantly, we utilized two phenotypically diverse strains that allowed us to identify biofilm-specific gene co-expression networks. One of these networks was highlighted by a gene encoding a ceramide synthase, designated barA, with a striking increase in barA transcript abundance specifically in the biofilm. Null mutants of barA in two strain backgrounds display a stunted biofilm morphology, with some strain-specific differences in the impact of biofilm biomass. Importantly, barA has a role in regulating susceptibility to the ergosterol-targeting antifungal drugs voriconazole and amphotericin B. These data identify biofilm-specific genes in A. fumigatus for further study and highlight the importance of fungal ceramide synthases in mediating antifungal drug susceptibility in infection-relevant biofilms. IMPORTANCE:Biofilms are problematic structures in the context of microbial infections due to their ability to resist both host- and drug-mediated attempts at tissue sterilization. Consequently, it is imperative to identify mechanisms underlying the development of these structures and the emergent properties they develop. The filamentous fungal pathogen Aspergillus fumigatus forms robust-structured biofilms that are resistant to contemporary antifungal drug treatments, although the mechanisms are ill-defined. In this study, we compared the transcriptional landscape of two A. fumigatus reference strains grown as biofilms and in planktonic culture conditions to identify biofilm-specific genes and pathways. These analyses and subsequent genetic and phenotypic studies revealed that a ceramide synthase is important for biofilm development and is involved in antifungal drug susceptibility of the biofilm. Consequently, these data support the rationale for targeting fungal lipid homeostasis for antifungal therapeutic development, particularly in the context of biofilm-mediated infections.

Background Elexacaftor/tezacaftor/ivacaftor (ETI) has impacted the ability for people with cystic fibrosis (PwCF) to spontaneously expectorate sputum, leading to lower respiratory sampling rates and infection detection challenges. Home sampling may permit a potential strategy for fungal detection in PwCF. Methods We conducted a prospective decentralised cohort study of PwCF to test the feasibility of home sputum collection and ambient temperature transport for Aspergillus fumigatus (Af) detection. Participants collected and shipped weekly sputum samples from home to the laboratory for fungal culture and completed electronic questionnaires. Descriptive statistics were calculated for patient factors, sputum characteristics and Af-positive cultures. We used a generalised estimating equations model to determine the association between highly effective modulator therapy (HEMT) and sputum volume. Results We enrolled 76 adults with cystic fibrosis (CF) with a median (interquartile range) forced expiratory volume in 1 s (FEV1) % predicted of 72.5% (53.8–86.3). 60 (79%) were on ETI and 44 (58%) had a history of Aspergillus. 70 (92%) successfully collected and shipped three or more sputum samples. Of 284 samples received, 83% arrived within one day. Sputum collection was reported as easy in 83 (29%) and somewhat easy in 114 (40%) collection events. Sputum volume from PwCF on HEMT was 36% lower than those not on HEMT (36%, 95% CI 3–58; p=0.03), adjusting for covariates. Af was detected in 205 (73%) of home sputum samples. Conclusion Home sputum collection is feasible in adults with CF. Af was detected in remotely collected sputum samples. Further work to assess the validity of home sputum samples in PwCF is necessary to determine the value of remote specimens in clinical and research settings.

Fungal keratitis is a severe corneal infection most commonly caused by filamentous fungi. Even with prompt treatment with current antifungals, it often results in corneal perforation and blindness. In this report, we observe that the beta-adrenergic antagonist, propranolol, displays antifungal activity against Aspergillus and Fusarium corneal isolates in vitro and strikingly blocks disease establishment in a murine model of Aspergillus fumigatus keratitis. These findings suggest that beta-blockers have potential as a novel FK treatment.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT00997035 (MUTT Trial).

Two of the three most commonly used classes of antifungal drugs target the fungal membrane through perturbation of sterol biosynthesis or function. In addition to these triazole and polyene antifungals, recent research is identifying new antifungal molecules that perturb lipid biosynthesis and function. Here, we review fungal lipid biosynthesis pathways and their potential as targets for antifungal drug development. An emerging goal is discovering new molecules that potentiate contemporary antifungal drugs in part through perturbation of lipid form and function.

Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in a broad spectrum of individuals. Diagnosis and treatment of aspergillosis often occur during stages of infection when A. fumigatus has formed dense networks of hyphae within the lung. These dense hyphal networks are multicellular, encased in a layer of extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, characteristics which are defining features of a microbial biofilm. A mode of growth similar to these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture model. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized an RNA sequencing approach to evaluate changes in transcript levels during A. fumigatus biofilm development. These analyses revealed an increase in transcripts associated with fermentation and a concomitant decrease in oxidative phosphorylation related transcripts. Further investigation revealed that ethanol and butanediol fermentation is important for mature biofilm biomass maintenance. Correspondingly, a gene (silG), a predicted transcription factor, was observed to also be required for mature biofilm biomass maintenance. Taken together, these data suggest temporal changes in A. fumigatus metabolism during biofilm development are required to maintain a fully mature biofilm. IMPORTANCE:Aspergillus fumigatus is the most common etiological agent of a collection of diseases termed aspergillosis. Invasive P ulmonary A spergillosis (IPA), a severe form of aspergillosis, is highlighted by invasive growth of fungal hyphae into host lung tissue. Strains that are susceptible to antifungal therapies in vitro frequently fail to respond to treatment in vivo, resulting in high mortality rates even with treatment. It is now appreciated that this decreased antifungal efficacy in vivo is, in part, likely due to biofilm-like growth of the fungus. A. fumigatus biofilms have been shown to develop regions of limited oxygen availability that are hypothesized to induce cell quiescence and drug resistance. Understanding the mechanisms by which A. fumigatus induces, develops, and maintains biofilms to evade antifungal therapies is expected to illuminate biofilm-specific therapeutic targets. Here we present transcriptomics data of developing A. fumigatus biofilms and from these data define genes related to fungal fermentation and regulation of transcription important for maintenance of mature A. fumigatus biofilms.

Fungal infections are difficult to prevent and treat in large part due to strain heterogeneity, which confounds diagnostic predictability. Yet, the genetic mechanisms driving strain-to-strain variation remain poorly understood. Here, we determined the extent to which Starships—giant transposons capable of mobilizing numerous fungal genes—generate genetic and phenotypic variability in the opportunistic human pathogen Aspergillus fumigatus. We analyzed 519 diverse strains, including 11 newly sequenced with long-read technology and multiple isolates of the same reference strain, to reveal 20 distinct Starships that are generating genomic heterogeneity over timescales relevant for experimental reproducibility. Starship-mobilized genes encode diverse functions, including known biofilm-related virulence factors and biosynthetic gene clusters, and many are differentially expressed during infection and antifungal exposure in a strain-specific manner. These findings support a new model of fungal evolution wherein Starships help generate variation in genome structure, gene content, and expression among fungal strains. Together, our results demonstrate that Starships are a previously hidden mechanism generating genotypic and, in turn, phenotypic heterogeneity in a major human fungal pathogen.IMPORTANCENo “one size fits all” option exists for treating fungal infections in large part due to genetic and phenotypic variability among strains. Accounting for strain heterogeneity is thus fundamental for developing efficacious treatments and strategies for safeguarding human health. Here, we report significant progress toward achieving this goal by uncovering a previously hidden mechanism generating heterogeneity in the human fungal pathogen Aspergillus fumigatus: giant transposons, called Starships, that span dozens of kilobases and mobilize fungal genes as cargo. By conducting a systematic investigation of these unusual transposons in a single fungal species, we demonstrate their contributions to population-level variation at the genome, pangenome, and transcriptome levels. The Starship compendium we develop will not only help predict variation introduced by these elements in laboratory experiments but will serve as a foundational resource for determining how Starships impact clinically relevant phenotypes, such as antifungal resistance and pathogenicity.

Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in humans. Diagnosis and treatment of aspergillosis often occur when A. fumigatus has formed dense networks of hyphae within the lung. These hyphal networks are multicellular, encased in an extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, similar to bacterial biofilms. A model of these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized RNA sequencing, in silico metabolic modeling, and molecular genetics approaches to identify A. fumigatus genes and metabolic pathways critical for biofilm development. These analyses revealed that ethanol and butanediol fermentation pathways are important for the development of a mature A. fumigatus biofilm. Correspondingly, a predicted transcription factor (silG) was observed to be required for mature biofilm development. Taken together, these data define key genes and metabolic pathways critical for A. fumigatus biofilm development. IMPORTANCE:Aspergillus fumigatus is the most common etiological agent of a collection of diseases termed aspergillosis. Chronic and invasive manifestations of aspergillosis are highlighted by the development of biofilm-like structures on and in tissue. These biofilm structures are resistant to contemporary antifungal drugs, even for strains that are susceptible by standard antimicrobial susceptibility testing methods. Consequently, understanding the mechanisms by which A. fumigatus induces, develops, and maintains biofilms to evade antifungal therapies is expected to illuminate biofilm-specific therapeutic targets. Here, we identify genes involved in fungal fermentation and regulation of transcription as important mediators of A. fumigatus biofilm development.

SUMMARY Advances in modern medical therapies for many previously intractable human diseases have improved patient outcomes. However, successful disease treatment outcomes are often prevented due to invasive fungal infections caused by the environmental mold Aspergillus fumigatus . As contemporary antifungal therapies have not experienced the same robust advances as other medical therapies, defining mechanisms of A. fumigatus disease initiation and progression remains a critical research priority. To this end, the World Health Organization recently identified A. fumigatus as a research priority human fungal pathogen and the Centers for Disease Control has highlighted the emergence of triazole-resistant A. fumigatus isolates. The expansion in the diversity of host populations susceptible to aspergillosis and the complex and dynamic A. fumigatus genotypic and phenotypic diversity call for a reinvigorated assessment of aspergillosis pathobiological and drug-susceptibility mechanisms. Here, we summarize recent advancements in the field and discuss challenges in our understanding of A. fumigatus heterogeneity and its pathogenesis in diverse host populations.

Inflection points are pivotal moments immediately preceding self-directed violence (SDV; i.e., self-injury and suicide). This study qualitatively examined factors that contributed to halting SDV during inflection points. Participants (N = 166) completing an online survey were community-dwelling adults in the United Kingdom with some form of SDV lived experience. Thematic analysis yielded the following results. The most common themes across SDV inflection points were (in descending order): concern for the negative impact on others (e.g., fear of hurting loved ones), use of adaptive coping methods (e.g., general use of coping skills), physical deterrents (e.g., scarring, pain), social contact (sense of connection in the moment), and concern for negative consequences on oneself (e.g., fear of punishment). Healthy coping skill use contributed to halting both self-injury and suicide. Self-injury inflection points were largely characterized by intrapersonal factors, whereas suicide inflection points were highly interpersonal in nature. Implications for clinical practice, theory, and research are discussed.

Background: Hate-motivated behaviour (HMB) ranges from microaggressions to criminal acts and is a public health concern with wide-ranging consequences. Aims: The current study aimed to examine the mental health correlates of HMB perpetration, victimisation and co-occurring victimisation/perpetration. Methods: Participants ( n = 447) completed an online cross-sectional survey assessing demographic factors, HMB (perpetration and victimisation), positive mental wellbeing and symptoms of depression and anxiety. Results: HMB victimisation was associated with lower positive mental wellbeing and increased symptoms of anxiety and depression. However, neither HMB perpetration nor co-occurring perpetration/victimisation were associated with any of the three mental health outcome measures. Conclusion: Experiencing HMB as a victim is linked to increased psychological distress. Additional research, which focuses on sampling populations who are known to be at greater risk for involvement in HMB, is needed to fully understand the impact of the victim-offender overlap on mental health outcomes.

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, patients who were treated with BTKi, 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 patients who are susceptible.

Members of the lesbian, gay, bisexual, transgender, queer, and other gender and sexual minority (LGBTQ+) community are disproportionately affected by illicit substance use, yet sexual orientation and gender identity are largely omitted from criminology frameworks. LGBTQ+ identity is incorporated into Akers' Social Structure-Social Learning (SSSL) model to suggest that existing disparate substance use patterns may be attributed to variation in substance-related definitions, peer models, and reinforcement. Data from 2,349 young adults were used to estimate structural equations models. LGBTQ+ respondents reported greater substance use than peers. Consistent with theoretical expectations, this relationship was fully mediated by social learning constructs. This study justifies incorporating sexual orientation and gender identity into the SSSL model and suggests peer-based substance use interventions are particularly influential within the LGBTQ+ community.

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.

Aspergillus fumigatus is a filamentous fungus abundant in the environment and the most common causative agent of a spectrum of human diseases collectively termed aspergillosis. Invasive pulmonary aspergillosis is caused by deficiencies in innate immune function that result in the inability of the host to clear inhaled Aspergillus conidia that then germinate and form invasive hyphae. Myeloid cells, and their ability to generate reactive oxygen species (ROS), are essential for conidia clearance from the host. To combat ROS, A. fumigatus employs an expansive antioxidant system, though how these canonical antioxidant mechanisms contribute to infection initiation and disease progression remain to be fully defined. Recent research has identified noncanonical pathways in the A. fumigatus ROS response and new host populations with ROS deficiencies that are at-risk for invasive aspergillosis. Here, we highlight recent developments in the understanding of ROS at the interface of the dynamic A. fumigatus-host interaction.