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.

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.

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.

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.

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.

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.

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.

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.

Antifungal Resistance in Pulmonary Aspergillosis.

Aspergilli may cause various pulmonary diseases in humans, including allergic bronchopulmonary aspergillosis (ABPA), chronic pulmonary aspergillosis (CPA), and acute invasive pulmonary aspergillosis (IPA). In addition, chronic colonization may occur in cystic fibrosis (CF). Aspergillus fumigatus represents the main pathogen, which may employ different morphotypes, for example, conidia, hyphal growth, and asexual sporulation, in the various Aspergillus diseases. These morphotypes determine the ease by which A. fumigatus can adapt to stress by antifungal drug exposure, usually resulting in one or more resistance mutations. Key factors that enable the emergence of resistance include genetic variation and selection. The ability to create genetic variation depends on the reproduction mode, including, sexual, parasexual, and asexual, and the population size. These reproduction cycles may take place in the host and/or in the environment, usually when specific conditions are present. Environmental resistance is commonly characterized by tandem repeat (TR)-mediated mutations, while in-host resistance selection results in single-resistance mutations. Reported cases from the literature indicate that environmental resistance mutations are almost exclusively present in patients with IA indicating that the risk for in-host resistance selection is very low. In aspergilloma, single-point mutations are the dominant resistance genotype, while in other chronic Aspergillus diseases, for example, ABPA, CPA, and CF, both TR-mediated and single-resistance mutations are reported. Insights into the pathogenesis of resistance selection in various Aspergillus diseases may help to improve diagnostic and therapeutic strategies.

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.

Proteome-Wide Analysis of Lysine 2-Hydroxyisobutyrylation in Aspergillus fumigatus.

Aspergillus fumigatus is the significant causative agent in cases of invasive aspergillosis, leading to a high mortality rate in immunocompromised patients. A comprehensive understanding of its growth patterns and metabolic processes within the host is a critical prerequisite for the development of effective antifungal strategies. Lysine 2-hydroxyisobutyrylation (Khib) is a highly conserved protein posttranslational modifications (PTM) found in various organisms. In this study, we investigate the biological impact of Khib in A. fumigatus. Using a combination of antibody enrichment with the conventional LC-MS/MS method, the pattern of Khib-modification in proteins and their respective sites were analyzed in a wild type strain of A. fumigatus. Our findings revealed 3494 Khib-modified proteins with a total of 18,091 modified sites in this strain. Functional enrichment analysis indicated that these Khib-modified proteins participate in a diverse range of cellular functions, spanning various subcellular locations such as ribosome biosynthesis, protein synthesis and nucleocytoplasmic transport. Notably, when compared with other reported eukaryotes, A. fumigatus exhibited consistently higher numbers of Khib-modified proteins, suggesting the potential significance of this modification in this organism. An interesting observation is the prevalence of Khib modifications in most enzymes involved in the ergosterol synthesis pathway. The insights gathered from this study provide new avenue for studying PTM-associated mechanisms in fungal growth and offer potential implication for antifungal drug development.

Functional, transcriptomic, and lipidomic studies of the choC gene encoding a phospholipid methyltransferase in Aspergillus fumigatus.

IMPORTANCE: This study explored the phospholipid metabolic pathway in A. fumigatus and its relationship with fungal growth, metabolism, and pathogenicity. ChoC, based on its critical roles in many aspects of the fungus and relatively conserved characteristics in filamentous fungi with low similarity with mammalian ones, can be a novel target of new antifungal drugs.

TRAF3 gene regulates macrophage migration and activation by lung epithelial cells infected with Aspergillus fumigatus.

IMPORTANCE: Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

Aspergillosis in a colony of Humboldt penguins (Spheniscus humboldti) in a french zoological park: evaluation of environmental exposure.

Aspergillosis is a major health problem in captive penguins due to the inhalation and the development of airborne spores of opportunistic environmental molds of the genus Aspergillus. Diagnosis is often delayed and treatments, based on the use of azole antifungals, are not fully effective. This study assesses the risk of exposure to Aspergillus sp. and determines the environmental reservoirs in the direct environment of a colony of Humboldt penguins (Spheniscus humboldti) in a zoological park in Paris, and the risk of contamination with resistant isolates. Every 15 days between February and May 2022, environmental samples (air and subtract from the nests, pond water, pigeon and penguin droppings) were carried out in the penguin enclosure as well as clinical samples (one-time non-invasive sampling on chicks), and screened for Aspergillus sp. conidia. From 191 environmental samples, 264 strains of Aspergillus including 221 strains of A. fumigatus were isolated, mostly from ambient air, in the nests, and pond water. No "at risk" areas in the penguin environment have been highlighted, nor an increased risk because of the proximity with urban wild birds. However, the load of airborne Aspergillus in the nests increased significantly with outdoor temperature. Of the 221 strains isolated, we identified only one azole-resistant strain, displaying the TR34/L98H mutation in the cyp51A gene. This low prevalence of resistant strains may probably be partly explained by the urban location of the zoological park, surrounded by kilometers of urban areas without agricultural activities.

Aspergillus fumigatus strains that evolve resistance to the agrochemical fungicide ipflufenoquin in vitro are also resistant to olorofim.

Widespread use of azole antifungals in agriculture has been linked to resistance in the pathogenic fungus Aspergillus fumigatus. We show that exposure of A. fumigatus to the agrochemical fungicide, ipflufenoquin, in vitro can select for strains that are resistant to olorofim, a first-in-class clinical antifungal with the same mechanism of action. Resistance is caused by non-synonymous mutations within the target of ipflufenoquin/olorofim activity, dihydroorotate dehydrogenase (DHODH), and these variants have no overt growth defects.

Species-specific PCR primers for simultaneous detection of Aspergillus fumigatus, Aspergillus terreus, Candida albicans and Candida glabrata in invasive fungal infections.

A rapid and accurate diagnosis of invasive fungal infections (IFIs) has been a great challenge particularly in cases requiring prompt antifungal treatment. In this study, four primer pairs were designed for a quadruplex PCR assay, which was developed for detection of four fungal species simultaneously. DNA extraction of cultured colonies and spiked blood samples were performed using conventional (phenol-chloroform) techniques and commercial DNA extraction kit. The optimum annealing temperature for this assay was 60°C. The assay was able to amplify all four genes and showed 100% specificity. No amplification of any genes was obtained against other species (n=14), which included two bacteria species. In conclusion, this quadruplex PCR assay is specific, rapid and reliable to detect A. fumigatus, A. terreus, C. albicans and C. glabrata simultaneously.

The MYST Family Histone Acetyltransferase SasC Governs Diverse Biological Processes in Aspergillus fumigatus.

The conserved MYST proteins form the largest family of histone acetyltransferases (HATs) that acetylate lysines within the N-terminal tails of histone, enabling active gene transcription. Here, we have investigated the biological and regulatory functions of the MYST family HAT SasC in the opportunistic human pathogenic fungus Aspergillus fumigatus using a series of genetic, biochemical, pathogenic, and transcriptomic analyses. The deletion (Δ) of sasC results in a drastically reduced colony growth, asexual development, spore germination, response to stresses, and the fungal virulence. Genome-wide expression analyses have revealed that the ΔsasC mutant showed 2402 significant differentially expressed genes: 1147 upregulated and 1255 downregulated. The representative upregulated gene resulting from ΔsasC is hacA, predicted to encode a bZIP transcription factor, whereas the UV-endonuclease UVE-1 was significantly downregulated by ΔsasC. Furthermore, our Western blot analyses suggest that SasC likely catalyzes the acetylation of H3K9, K3K14, and H3K29 in A. fumigatus. In conclusion, SasC is associated with diverse biological processes and can be a potential target for controlling pathogenic fungi.