The sterol C-24 methyltransferase encoding gene, erg6, is essential for viability of Aspergillus species.

Triazoles, the most widely used class of antifungal drugs, inhibit the biosynthesis of ergosterol, a crucial component of the fungal plasma membrane. Inhibition of a separate ergosterol biosynthetic step, catalyzed by the sterol C-24 methyltransferase Erg6, reduces the virulence of pathogenic yeasts, but its effects on filamentous fungal pathogens like Aspergillus fumigatus remain unexplored. Here, we show that the lipid droplet-associated enzyme Erg6 is essential for the viability of A. fumigatus and other Aspergillus species, including A. lentulus, A. terreus, and A. nidulans. Downregulation of erg6 causes loss of sterol-rich membrane domains required for apical extension of hyphae, as well as altered sterol profiles consistent with the Erg6 enzyme functioning upstream of the triazole drug target, Cyp51A/Cyp51B. Unexpectedly, erg6-repressed strains display wild-type susceptibility against the ergosterol-active triazole and polyene antifungals. Finally, we show that erg6 repression results in significant reduction in mortality in a murine model of invasive aspergillosis. Taken together with recent studies, our work supports Erg6 as a potentially pan-fungal drug target.

First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

BACKGROUND: Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. OBJECTIVES: This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. MATERIALS AND METHODS: Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. RESULTS: Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. CONCLUSIONS: These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

Peptide Mimics of the Cysteine-Rich Regions of HapX and SreA Bind a [2Fe-2S] Cluster In Vitro.

HapX and SreA are transcription factors that regulate the response of the fungus Aspergillus fumigatus to the availability of iron. During iron starvation, HapX represses genes involved in iron consuming pathways and upon a shift to iron excess, HapX activates these same genes. SreA blocks the expression of genes needed for iron uptake during periods of iron availability. Both proteins possess cysteine-rich regions (CRR) that are hypothesized to be necessary for the sensing of iron levels. However, the contribution of each of these domains to the function of the protein has remained unclear. Here, the ability of peptide analogs of each CRR is determined to bind an iron-sulfur cluster in vitro. UV-vis and resonance Raman (RR) spectroscopies reveal that each CRR is capable of coordinating a [2Fe-2S] cluster with comparable affinities. The iron-sulfur cluster coordinated to the CRR-B domain of HapX displays particularly high stability. The data are consistent with HapX and SreA mediating responses to cellular iron levels through the direct coordination of [2Fe-2S] clusters. The high stability of the CRR-B peptide may also find use as a starting point for the development of new green catalysts.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Discovery of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in Aspergillus fumigatus mycelium.

The cellular surface of the pathogenic filamentous fungus Aspergillus fumigatus is enveloped in a mannose layer, featuring well-established fungal-type galactomannan and O-mannose-type galactomannan. This study reports the discovery of cell wall component in A. fumigatus mycelium, which resembles N-glycan outer chains found in yeast. The glycosyltransferases involved in its biosynthesis in A. fumigatus were identified, with a focus on two key α-(1→2)-mannosyltransferases, Mnn2 and Mnn5, and two α-(1→6)-mannosyltransferases, Mnn9 and Van1. In vitro examination revealed the roles of recombinant Mnn2 and Mnn5 in transferring α-(1→2)-mannosyl residues. Proton nuclear magnetic resonance (1H-NMR) analysis of cell wall extracts from the ∆mnn2∆mnn5 strain indicated the existence of an α-(1→6)-linked mannan backbone in the A. fumigatus mycelium, with Mnn2 and Mnn5 adding α-(1→2)-mannosyl residues to this backbone. The α-(1→6)-linked mannan backbone was absent in strains where mnn9 or van1 was disrupted in the parental ∆mnn2∆mnn5 strain in A. fumigatus. Mnn9 and Van1 functioned as α-(1→6)-linked mannan polymerases in heterodimers when co-expressed in Escherichia coli, indicating their crucial role in biosynthesizing the α-(1→6)-linked mannan backbone. Disruptions of these mannosyltransferases did not affect fungal-type galactomannan biosynthesis. This study provides insights into the complexity of fungal cell wall architecture and a better understanding of mannan biosynthesis in A. fumigatus. IMPORTANCE: This study unravels the complexities of mannan biosynthesis in A. fumigatus, a key area for antifungal drug discovery. It reveals the presence of α-(1→6)-linked mannan structures resembling yeast N-glycan outer chains in A. fumigatus mycelium, offering fresh insights into the fungal cell wall's design. Key enzymes, Mnn2, Mnn5, Mnn9, and Van1, are instrumental in this process, with Mnn2 and Mnn5 adding specific mannose residues and Mnn9 and Van1 assembling the α-(1→6)-linked mannan structures. Although fungal-type galactomannan's presence in the cell wall is known, the existence of an α-(1→6)-linked mannan adds a new dimension to our understanding. This intricate web of mannan biosynthesis opens avenues for further exploration and enhances our understanding of fungal cell wall dynamics, paving the way for targeted drug development.

The EH domain-containing protein, EdeA, is involved in endocytosis, cell wall integrity, and pathogenicity in Aspergillus fumigatus.

Endocytosis has been extensively studied in yeasts, where it plays crucial roles in growth, signaling regulation, and cell-surface receptor internalization. However, the biological functions of endocytosis in pathogenic filamentous fungi remain largely unexplored. In this study, we aimed to functionally characterize the roles of EdeA, an ortholog of the Saccharomyces cerevisiae endocytic protein Ede1, in Aspergillus fumigatus. EdeA was observed to be distributed as patches on the plasma membrane and concentrated in the subapical collar of hyphae, a localization characteristic of endocytic proteins. Loss of edeA caused defective hyphal polarity, reduced conidial production, and fewer sites of endocytosis initiations than that of the parental wild type. Notably, the edeA null mutant exhibited increased sensitivity to cell wall-disrupting agents, indicating a role for EdeA in maintaining cell wall integrity in A. fumigatus. This observation was further supported by the evidence showing that the thickness of the cell wall in the ΔedeA mutant increased, accompanied by abnormal activation of MpkA, a key component in the cell wall integrity pathway. Additionally, the ΔedeA mutant displayed increased pathogenicity in the Galleria mellonella wax moth infection model, possibly due to alterations in cell wall morphology. Site-directed mutagenesis identified the conserved residue E348 within the third EH (Eps15 homology) domain of EdeA as crucial for its subcellular localization and functions. In conclusion, our results highlight the involvement of EdeA in endocytosis, hyphal polarity, cell wall integrity, and pathogenicity in A. fumigatus. IMPORTANCE: Aspergillus fumigatus is a significant human pathogenic fungus known to cause invasive aspergillosis, a disease with a high mortality rate. Understanding the basic principles of A. fumigatus pathogenicity is crucial for developing effective strategies against this pathogen. Previous research has underscored the importance of endocytosis in the infection capacity of pathogenic yeasts; however, its biological function in pathogenic mold remains largely unexplored. Our characterization of EdeA in A. fumigatus sheds light on the role of endocytosis in the development, stress response, and pathogenicity of pathogenic molds. These findings suggest that the components of the endocytosis process may serve as potential targets for antifungal therapy.

A secondary mechanism of action for triazole antifungals in Aspergillus fumigatus mediated by hmg1.

Triazole antifungals function as ergosterol biosynthesis inhibitors and are frontline therapy for invasive fungal infections, such as invasive aspergillosis. The primary mechanism of action of triazoles is through the specific inhibition of a cytochrome P450 14-α-sterol demethylase enzyme, Cyp51A/B, resulting in depletion of cellular ergosterol. Here, we uncover a clinically relevant secondary mechanism of action for triazoles within the ergosterol biosynthesis pathway. We provide evidence that triazole-mediated inhibition of Cyp51A/B activity generates sterol intermediate perturbations that are likely decoded by the sterol sensing functions of HMG-CoA reductase and Insulin-Induced Gene orthologs as increased pathway activity. This, in turn, results in negative feedback regulation of HMG-CoA reductase, the rate-limiting step of sterol biosynthesis. We also provide evidence that HMG-CoA reductase sterol sensing domain mutations previously identified as generating resistance in clinical isolates of Aspergillus fumigatus partially disrupt this triazole-induced feedback. Therefore, our data point to a secondary mechanism of action for the triazoles: induction of HMG-CoA reductase negative feedback for downregulation of ergosterol biosynthesis pathway activity. Abrogation of this feedback through acquired mutations in the HMG-CoA reductase sterol sensing domain diminishes triazole antifungal activity against fungal pathogens and underpins HMG-CoA reductase-mediated resistance.

Genomic diversity of the pathogenic fungus Aspergillus fumigatus in Japan reveals the complex genomic basis of azole resistance.

Aspergillus fumigatus is a pathogenic fungus with a global distribution. The emergence of azole-resistant A. fumigatus (ARAf) other than the TR-mutants is a problem in Japan. Additionally, the genetic diversity of A. fumigatus strains in Japan remains relatively unknown. Here we show the diversity in the A. fumigatus strains isolated in Japan as well as the complexity in the global distribution of the pathogenic strains. First, we analyzed the genome sequences of 171 strains from Japan as well as the antifungal susceptibility of these strains. Next, we conducted a population analysis of 876 strains by combining the available genomic data for strains isolated worldwide, which were grouped in six clusters. Finally, a genome-wide association study identified the genomic loci associated with ARAf strains, but not the TR-mutants. These results highlight the complexity of the genomic mechanism underlying the emergence of ARAf strains other than the TR-mutants.

Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.

Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.

The S-S bridge mutation between the A2 and A4 loops (T416C-I432C) of Cel7A of Aspergillus fumigatus enhances catalytic activity and thermostability.

Disulfide bonds are important for maintaining the structural conformation and stability of the protein. The introduction of the disulfide bond is a promising strategy to increase the thermostability of the protein. In this report, cysteine residues are introduced to form disulfide bonds in the Glycoside Hydrolase family GH 7 cellobiohydrolase (GH7 CBHs) or Cel7A of Aspergillus fumigatus. Disulfide by Design 2.0 (DbD2), an online tool is used for the detection of the mutation sites. Mutations are created (D276C-G279C; DSB1, D322C-G327C; DSB2, T416C-I432C; DSB3, G460C-S465C; DSB4) inside and outside of the peripheral loops but, not in the catalytic region. The introduction of cysteine in the A2 and A4 loop of DSB3 mutant showed higher thermostability (70% activity at 70°C), higher substrate affinity (Km = 0.081 mM) and higher catalytic activity (Kcat = 9.75 min-1; Kcat/Km = 120.37 mM min-1) compared to wild-type AfCel7A (50% activity at 70°C; Km = 0.128 mM; Kcat = 4.833 min-1; Kcat/Km = 37.75 mM min-1). The other three mutants with high B factor showed loss of thermostability and catalytic activity. Molecular dynamic simulations revealed that the mutation T416C-I432C makes the tunnel wider (DSB3: 13.6 Å; Wt: 5.3 Å) at the product exit site, giving flexibility in the entrance region or mobility of the substrate in the exit region. It may facilitate substrate entry into the catalytic tunnel and release the product faster than the wild type, whereas in other mutants, the tunnel is not prominent (DSB4), the exit is lost (DSB1), and the ligand binding site is absent (DSB2). This is the first report of the gain of function of both thermostability and enzyme activity of cellobiohydrolase Cel7A by disulfide bond engineering in the loop.IMPORTANCEBioethanol is one of the cleanest renewable energy and alternatives to fossil fuels. Cost efficient bioethanol production can be achieved through simultaneous saccharification and co-fermentation that needs active polysaccharide degrading enzymes. Cellulase enzyme complex is a crucial enzyme for second-generation bioethanol production from lignocellulosic biomass. Cellobiohydrolase (Cel7A) is an important member of this complex. In this work, we engineered (disulfide bond engineering) the Cel7A to increase its thermostability and catalytic activity which is required for its industrial application.

Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole.

The emergence of azole-resistant Aspergillus fumigatus (ARAf) across the world is an important public health concern. We sought to determine if propiconazole, a demethylase inhibitor (DMI) fungicide, exerted a selective pressure for ARAf in a tomato production environment following multiple exposures to the fungicide. A tomato field trial was established in 2019 and propiconazole was applied weekly until harvest. Soil, leaf, and fruit (when present) samples were collected at baseline and after each propiconazole application. A. fumigatus isolates (n, 178) were recovered and 173 were tested for susceptibility to itraconazole, posaconazole, voriconazole, and propiconazole in accordance with CLSI M38 guidelines. All the isolates were susceptible to medical triazoles and the propiconazole MIC ranged from 0.25 to 8 mg/L. A linear regression model was fitted that showed no longitudinal increment in the log2-fold azole MIC of the isolates collected after each propiconazole exposure compared to the baseline isolates. AsperGenius real-time multiplex assay ruled out TR34/L98H and TR46/Y121F/T289A cyp51A resistance markers in these isolates. Sequencing of a subset of isolates (n, 46) demonstrated widespread presence of F46Y/M172V/E427K and F46Y/M172V/N248T/D255E/E427K cyp51A mutations previously associated with reduced susceptibility to triazoles. IMPORTANCE: The agricultural use of azole fungicides to control plant diseases has been implicated as a major contributor to ARAf infections in humans. Our study did not reveal imposition of selection pressure for ARAf in a vegetable production system. However, more surveillance studies for ARAf in food crop production and other environments are warranted in understanding this public and One Health issue.

Protein disulfide isomerase 1 is required for RodA assembling-based conidial hydrophobicity of Aspergillus fumigatus.

The hydrophobic layer of Aspergillus conidia, composed of RodA, plays a crucial role in conidia transfer and immune evasion. It self-assembles into hydrophobic rodlets through intramolecular disulfide bonds. However, the secretory process of RodA and its regulatory elements remain unknown. Since protein disulfide isomerase (PDI) is essential for the secretion of many disulfide-bonded proteins, we investigated whether PDI is also involved in RodA secretion and assembly. By gene knockout and phenotypic analysis, we found that Pdi1, one of the four PDI-related proteins of Aspergillus fumigatus, determines the hydrophobicity and integrity of the rodlet layer of the conidia. Preservation of the thioredoxin-active domain of Pdi1 was sufficient to maintain conidial hydrophobicity, suggesting that Pdi1 mediates RodA assembly through its disulfide isomerase activity. In the absence of Pdi1, the disulfide mismatch of RodA in conidia may prevent its delivery from the inner to the outer layer of the cell wall for rodlet assembly. This was demonstrated using a strain expressing a key cysteine-mutated RodA. The dormant conidia of the Pdi1-deficient strain (Δpdi) elicited an immune response, suggesting that the defective conidia surface in the absence of Pdi1 exposes internal immunogenic sources. In conclusion, Pdi1 ensures the correct folding of RodA in the inner layer of conidia, facilitating its secretion into the outer layer of the cell wall and allowing self-assembly of the hydrophobic layer. This study has identified a regulatory element for conidia rodlet assembly.IMPORTANCEAspergillus fumigatus is the major cause of invasive aspergillosis, which is mainly transmitted by the inhalation of conidia. The spread of conidia is largely dependent on their hydrophobicity, which is primarily attributed to the self-assembly of the hydrophobic protein RodA on the cell wall. However, the mechanisms underlying RodA secretion and transport to the outermost layer of the cell wall are still unclear. Our study identified a critical role for Pdi1, a fungal protein disulfide isomerase found in regulating RodA secretion and assembly. Inhibition of Pdi1 prevents the formation of correct S-S bonds in the inner RodA, creating a barrier to RodA delivery and resulting in a defective hydrophobic layer. Our findings provided insight into the formation of the conidial hydrophobic layer and suggested potential drug targets to inhibit A. fumigatus infections by limiting conidial dispersal and altering their immune inertia.

Histone acetyltransferase Sas3 contributes to fungal development, cell wall integrity, and virulence in Aspergillus fumigatus.

Histone acetyltransferase (HAT)-mediated epigenetic modification is essential for diverse cellular processes in eukaryotes. However, the functions of HATs in the human pathogen Aspergillus fumigatus remain poorly understood. In this study, we characterized the functions of MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST)-family histone acetyltransferase something about silencing (Sas3) in A. fumigatus. Phenotypic analysis revealed that loss of Sas3 results in significant impairments in colony growth, conidiation, and virulence in the Galleria mellonella model. Subcellular localization and Western blot analysis demonstrated that Sas3 localizes to nuclei and is capable of acetylating lysine 9 and 14 of histone H3 in vivo. Importantly, we found that Sas3 is critical for the cell wall integrity (CWI) pathway in A. fumigatus as evidenced by hypersensitivity to cell wall-perturbing agents, altered cell wall thickness, and abnormal phosphorylation levels of CWI protein kinase MpkA. Furthermore, site-directed mutagenesis studies revealed that the conserved glycine residues G641 and G643 and glutamate residue E664 are crucial for the acetylation activity of Sas3. Unexpectedly, only triple mutations of Sas3 (G641A/G643A/E664A) displayed defective phenotypes similar to the Δsas3 mutant, while double or single mutations did not. This result implies that the role of Sas3 may extend beyond histone acetylation. Collectively, our findings demonstrate that MYST-family HAT Sas3 plays an important role in the fungal development, virulence, and cell wall integrity in A. fumigatus. IMPORTANCE: Epigenetic modification governed by HATs is indispensable for various cellular processes in eukaryotes. Nonetheless, the precise functions of HATs in the human pathogen Aspergillus fumigatus remain elusive. In this study, we unveil the roles of MYST-family HAT Sas3 in colony growth, conidiation, virulence, and cell wall stress response in A. fumigatus. Particularly, our findings demonstrate that Sas3 can function through mechanisms unrelated to histone acetylation, as evidenced by site-directed mutagenesis experiments. Overall, this study broadens our understanding of the regulatory mechanism of HATs in fungal pathogens.

The spatial organization of sphingofungin biosynthesis in Aspergillus fumigatus and its cross-interaction with sphingolipid metabolism.

Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.IMPORTANCEA balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.

Identifying genetic susceptibility to Aspergillus fumigatus infection using collaborative cross mice and RNA-Seq approach.

BACKGROUND: Aspergillus fumigatus (Af) is one of the most ubiquitous fungi and its infection potency is suggested to be strongly controlled by the host genetic background. The aim of this study was to search for candidate genes associated with host susceptibility to Aspergillus fumigatus (Af) using an RNAseq approach in CC lines and hepatic gene expression. METHODS: We studied 31 male mice from 25 CC lines at 8 weeks old; the mice were infected with Af. Liver tissues were extracted from these mice 5 days post-infection, and next-generation RNA-sequencing (RNAseq) was performed. The GENE-E analysis platform was used to generate a clustered heat map matrix. RESULTS: Significant variation in body weight changes between CC lines was observed. Hepatic gene expression revealed 12 top prioritized candidate genes differentially expressed in resistant versus susceptible mice based on body weight changes. Interestingly, three candidate genes are located within genomic intervals of the previously mapped quantitative trait loci (QTL), including Gm16270 and Stox1 on chromosome 10 and Gm11033 on chromosome 8. CONCLUSIONS: Our findings emphasize the CC mouse model's power in fine mapping the genetic components underlying susceptibility towards Af. As a next step, eQTL analysis will be performed for our RNA-Seq data. Suggested candidate genes from our study will be further assessed with a human cohort with aspergillosis.

A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus.

Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection (P < 0.001) and significantly improved 7-day survival (P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.IMPORTANCEInvasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillus-active antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) T-cell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine anti-Aspergillus CAR T-cell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibody-based CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR T-cell-based antifungal immunotherapy.

Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a.

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 102 copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

Electronic equipment and appliances in special wards of hospitals as a source of azole-resistant Aspergillus fumigatus: a multi-centre study from Iran.

BACKGROUND: Azole-resistant Aspergillus fumigatus (ARAf), reported as a global public health concern, has been unexpectedly observed in different countries. AIM: To identify ARAf and detect azole resistance related to the CYP51A mutation in different hospital environmental samples. METHODS: In this multi-centre study from Iran, surfaces of electronic equipment and appliances from different hospitals in Iran were sampled using cotton swabs. All samples were cultured using azole-containing agar plates (ACAPs). Recovered Aspergillus isolates were identified at the species level using partial DNA sequencing of the ß-tubulin gene. The azole susceptibility testing of A. fumigatus isolates was performed using the Clinical and Laboratory Standards Institute M38-A3 guideline. The sequencing of the CYP51A gene was also performed to detect mutations related to resistance. FINDINGS: Out of the 693 collected samples, 89 (12.8%) Aspergillus species were recovered from ACAPs. Aspergillus fumigatus (41.6%) was the most prevalent, followed by A. tubingensis (23.6%) and A. niger (15.6%). Among 37 isolates of A. fumigatus, 19 (51.3%) showed high minimum inhibitory concentration (MIC) values to at least one of the three azoles, voriconazole, itraconazole, and posaconazole. CYP51A polymorphisms were detected in all 19 isolates, of which 52.6% showed the TR34/L98H mutation. Other detected mutations were G432C, G448S, G54E/G138C, F46Y, and Y121F/M220I/D255E. T289F and G432C were the first reported mutations in ARAf. CONCLUSION: There was a considerable level of azole resistance in hospital environmental samples, a serious warning for patients vulnerable to aspergillosis. Our findings have also revealed a different mutation pattern in the CYP51A gene.

Rapid diagnosis of Aspergillus fumigatus endocarditis using mNGS assay: A case report and review of the literature.

Fungal endocarditis is caused mainly by Candida albicans and Aspergillus spp. and was first reported in the 1950s. Natural-valve endocarditis caused by Aspergillus is relatively uncommon. In this case, a 56-year-old male patient was admitted to the hospital on account of a cough accompanied by chills and fever and ineffective self-medication. Infective endocarditis was initially suspected based on echocardiography (indicating right atrial growth) and clinical manifestations. However, routine pathogen detections were always negative. The patient's condition was identified as Aspergillus fumigatus endocarditis (AFE) and was treated with targeted therapy, considering the detection of significant AFE sequences in the blood through metagenomic next-generation sequencing (mNGS). On this basis, the paper further summarizes the clinical manifestations, diagnosis, treatments, and outcomes of AFE endocarditis cases reported in recent years, aiming to provide a reference to better understand this rare infective disease and guide medical practitioners in choosing the right diagnostic and therapeutic strategy.