Human Fungal Pathogen Aspergillus Fumigatus Research Articles

Enhanced antifungal activity of siRNA-loaded anionic liposomes against the human pathogenic fungus Aspergillus fumigatus.

We developed siRNA-loaded anionic liposomes, co-encapsulating low-dose amphotericin B, to enhance siRNA penetration through the fungal cell wall of Aspergillus fumigatus. Targeting mRNAs of three key genes, these liposomes visibly inhibited fungal growth, demonstrating for the first time the antifungal potential of siRNA against human fungal pathogens.

Manipulation of host phagocytosis by fungal pathogens and therapeutic opportunities.

An important host defence mechanism against pathogens is intracellular killing, which is achieved through phagocytosis, a cellular process for engulfing and neutralizing extracellular particles. Phagocytosis results in the formation of matured phagolysosomes, which are specialized compartments that provide a hostile environment and are considered the end point of the degradative pathway. However, all fungal pathogens studied to date have developed strategies to manipulate phagosomal function directly and also indirectly by redirecting phagosomes from the degradative pathway to a non-degradative pathway with the expulsion and even transfer of pathogens between cells. Here, using the major human fungal pathogens Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans and Histoplasma capsulatum as examples, we discuss the processes involved in host phagosome-fungal pathogen interactions, with a focus on fungal evasion strategies. We also discuss recent approaches to targeting intraphagosomal pathogens, including the redirection of phagosomes towards degradative pathways for fungal pathogen eradication.

Adiponectin pathway activation dampens inflammation and enhances alveolar macrophage fungal killing via LC3-associated phagocytosis.

Although innate immunity is critical for antifungal host defense against the human opportunistic fungal pathogen Aspergillus fumigatus, potentially damaging inflammation must be controlled. Adiponectin (APN) is an adipokine produced mainly in adipose tissue that exerts anti-inflammatory effects in adipose-distal tissues such as the lung. We observed 100% mortality and increased fungal burden and inflammation in neutropenic mice with invasive aspergillosis (IA) that lack APN or the APN receptors AdipoR1 or AdipoR2. Alveolar macrophages (AMs), early immune sentinels that detect and respond to lung infection, express both receptors, and APN-/- AMs exhibited an inflammatory/M1 phenotype that was associated with decreased fungal killing. Pharmacological stimulation of AMs with AdipoR agonist AdipoRon partially rescued deficient killing in APN-/- AMs that was dependent on both receptors. Finally, APN-enhanced fungal killing was associated with increased activation of the non-canonical LC3 pathway of autophagy. Thus, our study identifies a novel role for APN in LC3-mediated killing of A. fumigatus.

Nitrate assimilation compensates for cell wall biosynthesis in the absence of Aspergillus fumigatus phosphoglucose isomerase.

Phosphoglucose isomerase (PGI) links glycolysis, the pentose phosphate pathway (PPP), and the synthesis of cell wall precursors in fungi by facilitating the reversible conversion between glucose-6-phosphate (Glc6p) and fructose-6-phosphate (Fru6P). In a previous study, we established the essential role of PGI in cell wall biosynthesis in the opportunistic human fungal pathogen Aspergillus fumigatus, highlighting its potential as a therapeutic target. In this study, we conducted transcriptomic analysis and discovered that the Δpgi mutant exhibited enhanced glycolysis, reduced PPP, and an upregulation of cell wall precursor biosynthesis pathways. Phenotypic analysis revealed defective protein N-glycosylation in the mutant, notably the absence of glycosylated virulence factors DPP V and catalase 1. Interestingly, the cell wall defects in the mutant were not accompanied by activation of the MpkA-dependent cell wall integrity (CWI) signaling pathway. Instead, nitrate assimilation was activated in the Δpgi mutant, stimulating glutamine synthesis and providing amino donors for chitin precursor biosynthesis. Blocking the nitrate assimilation pathway severely impaired the growth of the Δpgi mutant, highlighting the crucial role of nitrate assimilation in rescuing cell wall defects. This study unveils the connection between nitrogen assimilation and cell wall compensation in A. fumigatus.IMPORTANCEAspergillus fumigatus is a common and serious human fungal pathogen that causes a variety of diseases. Given the limited availability of antifungal drugs and increasing drug resistance, it is imperative to understand the fungus' survival mechanisms for effective control of fungal infections. Our previous study highlighted the essential role of A. fumigatus PGI in maintaining cell wall integrity, phosphate sugar homeostasis, and virulence. The present study further illuminates the involvement of PGI in protein N-glycosylation. Furthermore, this research reveals that the nitrogen assimilation pathway, rather than the canonical MpkA-dependent CWI pathway, compensates for cell wall deficiencies in the mutant. These findings offer valuable insights into a novel adaptation mechanism of A. fumigatus to address cell wall defects, which could hold promise for the treatment of infections.

Catching some air: a method to spatially quantify aerial triazole resistance in Aspergillus fumigatus.

Airborne triazole-resistant spores of the human fungal pathogen Aspergillus fumigatus are a significant human health problem as the agricultural use of triazoles has been selecting for cross-resistance to life-saving clinical triazoles. However, how to quantify exposure to airborne triazole-resistant spores remains unclear. Here, we describe a method for cost-effective wide-scale outdoor air sampling to measure both spore abundance as well as antifungal resistance fractions. We show that prolonged outdoor exposure of sticky seals placed in delta traps, when combined with a two-layered cultivation approach, can regionally yield sufficient colony-forming units (CFUs) for the quantitative assessment of aerial resistance levels at a spatial scale that was up to now unfeasible. When testing our method in a European pilot sampling 12 regions, we demonstrate that there are significant regional differences in airborne CFU numbers, and the triazole-resistant fraction of airborne spores is widespread and varies between 0 and 0.1 for itraconazole (∼4 mg/L) and voriconazole (∼2 mg/L). Our efficient and accessible air sampling protocol opens up extensive options for fine-scale spatial sampling and surveillance studies of airborne A. fumigatus.IMPORTANCEAspergillus fumigatus is an opportunistic fungal pathogen that humans and other animals are primarily exposed to through inhalation. Due to the limited availability of antifungals, resistance to the first choice class of antifungals, the triazoles, in A. fumigatus can make infections by this fungus untreatable and uncurable. Here, we describe and validate a method that allows for the quantification of airborne resistance fractions and quick genotyping of A. fumigatus TR-types. Our pilot study provides proof of concept of the suitability of the method for use by citizen-scientists for large-scale spatial air sampling. Spatial air sampling can open up extensive options for surveillance, health-risk assessment, and the study of landscape-level ecology of A. fumigatus, as well as investigating the environmental drivers of triazole resistance.

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

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

Beyond the Charge: Interplay of Nanogels' Functional Group and Zeta-Potential for Antifungal Drug Delivery to Human Pathogenic Fungus Aspergillus Fumigatus.

The ubiquitous mold Aspergillus fumigatus (A. fumigatus) is one of the main fungal pathogens causing invasive infections in immunocompromised humans. Conventional antifungal agents exhibit limited efficacy and often cause severe side effects. Nanoparticle-based antifungal delivery provides a promising alternative, which can increase local drug concentration; while, mitigating toxicity, thereby enhancing treatment efficacy. Previous research underscores the potential of poly(glycidol)-based nanogels (NG) with negative surface charge as carriers for delivering antifungals to A. fumigatus hyphae. In this study, NG is tailored with 2-carboxyethyl acrylate (CEA) or with phosphoric acid 2-hydroxyethyl acrylate (PHA). It is discovered that quenching with PHA clearly improves the adhesion of NG to hyphal surface and the internalization of NG into the hyphae under protein-rich conditions, surpassing the outcomes of non-quenched and CEA-quenched NG. This enhancement cannot be solely attributed to an increase in negative surface charge but appears to be contingent on the functional group of the quencher. Further, it is demonstrated that itraconazole-loaded, PHA-functionalized nanogels (NGxPHA-ITZ) show lower MIC in vitro and superior therapeutic effect in vivo against A. fumigatus compared to pure itraconazole. This confirms NGxPHA as a promising antifungal delivery system.

Fungal melanin suppresses airway epithelial chemokine secretion through blockade of calcium fluxing

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

Brilacidin, a host defense peptide mimetic, potentiates ibrexafungerp antifungal activity against the human pathogenic fungus Aspergillus fumigatus.

Invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. Aspergillus fumigatus causes a series of distinct invasive fungal infections have a high mortality rate causing more deaths annually than tuberculosis or malaria. A. fumigatus causes a spectrum of distinct clinical entities named aspergillosis, which the most severe form is the invasive pulmonary aspergillosis. There are few therapeutic options for treating aspergillosis and searching for new antifungal agents against this disease is very important. Here, we present brilacidin (BRI) as a synergizer o fibrexafungerp (IBX) against A. fumigatus. BRI is a small molecule host defense peptide mimetic that has previously exhibited broad-spectrum immunomodulatory/anti-inflammatory activity against bacteria and viruses. We propose the combination of BRI and IBX as a new antifungal combinatorial treatment against aspergillosis.

Variability in competitive fitness among environmental and clinical azole-resistant Aspergillus fumigatus isolates.

Azole resistance in the human fungal pathogen Aspergillus fumigatus presents a global public health challenge. Understanding the epidemic trends and evolutionary patterns of azole resistance is critical to prevent and control the spread of azole-resistant isolates. The primary cause is the mutation of the drug target 14α-sterol-demethylase Cyp51A, yet its impact on competitive ability remains uncertain. Our competition assays revealed a diverse range of fitness outcomes for environmental and clinical cyp51A-mutated isolates. We have shown that this fitness cost is not reliant on cyp51A mutations but might be linked to unknown mutations induced by stress conditions. Among these isolates, the majority displayed fitness costs, while a few displayed enhanced competitive ability, which may have a potential risk of spread and the need to closely monitor these isolates. Our observation reveals the variation in fitness costs among azole-resistant isolates of A. fumigatus, highlighting the significant role of fitness cost in the spread of resistant strains.

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.

Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus

ABSTRACT Asexual spores, called conidia, are key reproductive fungal particles that enable survival in harsh environmental conditions or host systems. The conidia can infect humans, animals, and plants to cause various fungal diseases. Transcription factors, including VosA, WetA, and SscA, have key roles in conidia formation and long-term survival in Aspergillus nidulans. Herein, we report the pleiotropic functions of SscA in the conidia of the human pathogen A. fumigatus. The deletion of sscA increased conidia formation despite decreased fungal growth. Absence of sscA impaired long-term survival and reduced spore resistance to various stresses, including heat, UV, and oxidation. Transcriptomic analyses showed that SscA involved the mRNA expression of cell wall organisation-related genes. Importantly, the sscA deletion mutant conidia contained an increased amount of β-glucan and chitin compared to wild type conidia. In addition, conidial gliotoxin production was decreased in the sscA deletion strain. Overall, SscA has pleiotropic roles in conidia formation, maturation and dormancy and mycotoxin production in A. fumigatus.

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.

The human fungal pathogen Aspergillus fumigatus can produce the highest known number of meiotic crossovers.

Sexual reproduction involving meiosis is essential in most eukaryotes. This produces offspring with novel genotypes, both by segregation of parental chromosomes as well as crossovers between homologous chromosomes. A sexual cycle for the opportunistic human pathogenic fungus Aspergillus fumigatus is known, but the genetic consequences of meiosis have remained unknown. Among other Aspergilli, it is known that A. flavus has a moderately high recombination rate with an average of 4.2 crossovers per chromosome pair, whereas A. nidulans has in contrast a higher rate with 9.3 crossovers per chromosome pair. Here, we show in a cross between A. fumigatus strains that they produce an average of 29.9 crossovers per chromosome pair and large variation in total map length across additional strain crosses. This rate of crossovers per chromosome is more than twice that seen for any known organism, which we discuss in relation to other genetic model systems. We validate this high rate of crossovers through mapping of resistance to the laboratory antifungal acriflavine by using standing variation in an undescribed ABC efflux transporter. We then demonstrate that this rate of crossovers is sufficient to produce one of the common multidrug resistant haplotypes found in the cyp51A gene (TR34/L98H) in crosses among parents harboring either of 2 nearby genetic variants, possibly explaining the early spread of such haplotypes. Our results suggest that genomic studies in this species should reassess common assumptions about linkage between genetic regions. The finding of an unparalleled crossover rate in A. fumigatus provides opportunities to understand why these rates are not generally higher in other eukaryotes.

Mitochondrial Membrane-Associated Protein Mba1 Confers Antifungal Resistance by Affecting the Production of Reactive Oxygen Species in Aspergillus fumigatus.

Azole resistance in the human fungal pathogen Aspergillus fumigatus is becoming a major threat to global health. To date, mutations in the azole target-encoding cyp51A gene have been implicated in conferring azole resistance, but a steady increase in the number of A. fumigatus isolates with azole resistance resulting from non-cyp51A mutations has been recognized. Previous studies have revealed that some isolates with non-cyp51A mutation-induced azole resistance are related to mitochondrial dysfunction. However, knowledge of the molecular mechanism underlying the involvement of non-cyp51A mutations is limited. In this study, using next-generation sequencing, we found that nine independent azole-resistant isolates without cyp51A mutations had normal mitochondrial membrane potential. Among these isolates, a mutation in a mitochondrial ribosome-binding protein, Mba1, conferred multidrug resistance to azoles, terbinafine, and amphotericin B but not caspofungin. Molecular characterization verified that the TIM44 domain of Mba1 was crucial for drug resistance and that the N terminus of Mba1 played a major role in growth. Deletion of mba1 had no effect on Cyp51A expression but decreased the fungal cellular reactive oxygen species (ROS) content, which contributed to mba1-mediated drug resistance. The findings in this study suggest that some non-cyp51A proteins drive drug resistance mechanisms that result from reduced ROS production induced by antifungals.

Surrogate infection model predicts optimal alveolar macrophage number for clearance of Aspergillus fumigatus infections

The immune system has to fight off hundreds of microbial invaders every day, such as the human-pathogenic fungus Aspergillus fumigatus. The fungal conidia can reach the lower respiratory tract, swell and form hyphae within six hours causing life-threatening invasive aspergillosis. Invading pathogens are continuously recognized and eliminated by alveolar macrophages (AM). Their number plays an essential role, but remains controversial with measurements varying by a factor greater than ten for the human lung. We here investigate the impact of the AM number on the clearance of A. fumigatus conidia in humans and mice using analytical and numerical modeling approaches. A three-dimensional to-scale hybrid agent-based model (hABM) of the human and murine alveolus allowed us to simulate millions of virtual infection scenarios, and to gain quantitative insights into the infection dynamics for varying AM numbers and infection doses. Since hABM simulations are computationally expensive, we derived and trained an analytical surrogate infection model on the large dataset of numerical simulations. This enables reducing the number of hABM simulations while still providing (i) accurate and immediate predictions on infection progression, (ii) quantitative hypotheses on the infection dynamics under healthy and immunocompromised conditions, and (iii) optimal AM numbers for combating A. fumigatus infections in humans and mice.

Anti-inflammatory and anti-biotic drug metronidazole loaded ZIF-90 nanoparticles as a pH responsive drug delivery system for improved pediatric sepsis management.

Sepsis is a life-threatening disease caused by the dis-functioning of the immune response to pathogenic infections. Despite, the discovery of modern therapeutics and treatments of sepsis are lacking due to the resistance of pathogens. Metronidazole is an antibiotic commonly used to treat bacterial infections, but usage is limited and challenging by a short half-life period. In this research work, fabricate a pH-responsive drug delivery system for controlled release of metronidazole targeted molecules. We exemplified that, the encapsulation of hydrophilic metronidazole drug within a hydrophobic ZIF-90 framework can be enhanced the pH-responsive drug release under acidic conditions. The ZIF-90 frameworks only decompose in under acidic solutions, they are highly stable in physiological conditions. The pH-responsive protonation mechanism of ZIF-90 frameworks promotes the quick release of metronidazole within cells. The antimicrobial proficiency of zinc and metronidazole will expose a synergistic effect in ROS-mediated bacterial inhibition and auto-immunity boosting of normal cells. In vitro, antibacterial activity results revealed that the MI@ZIF-90 nano drug delivery system effectively eradicated human infectious pathogens at the lowest concentrations. In anti-fungal activity, studies show excellent growth inhibition against human pathogenic fungi Aspergillus fumigatus and Candida albicans. Finally, the PBMC cytocompatibility study concludes, that the fabricated MI@ZIF-90 drug delivery system is non-toxic to biomedical applications. The overall research findings highlight the design of a smart drug delivery system for sepsis treatment. In future it will be an efficient, low-cost, and biocompatible pharmaceutics for pediatric sepsis management processes.

Aspergillus fumigatus hijacks human p11 to redirect fungal-containing phagosomes to non-degradative pathway

The decision whether endosomes enter the degradative or recycling pathway in mammalian cells is of fundamental importance for pathogen killing, and its malfunctioning has pathological consequences. We discovered that human p11 is a critical factor for this decision. The HscA protein present on the conidial surface of the human-pathogenic fungus Aspergillus fumigatus anchors p11 on conidia-containing phagosomes (PSs), excludes the PS maturation mediator Rab7, and triggers binding of exocytosis mediators Rab11 and Sec15. This reprogramming redirects PSs to the non-degradative pathway, allowing A.fumigatus to escape cells by outgrowth and expulsion as well as transfer of conidia between cells. The clinical relevance is supported by the identification of a single nucleotide polymorphism in the non-coding region of the S100A10 (p11) gene that affects mRNA and protein expression in response to A.fumigatus and is associated with protection against invasive pulmonary aspergillosis. These findings reveal the role of p11 in mediating fungal PS evasion.

Transcriptomics-based investigation of molecular mechanisms underlying synergistic antimicrobial effects of AgNPs and Domiphen on the human fungal pathogen Aspergillus fumigatus.

Critically ill patients have higher risk of serious fungal infections, such as invasive aspergillosis (IA) which is mainly caused by the human fungal pathogen Aspergillus fumigatus. Triazole drugs are the primary therapeutic agents for the first-line treatment of IA, which could easily cause drug resistance problems. Here, we assess the potential of AgNPs synthesized with Artemisia argyi leaf extract and domiphen as new antifungal agents to produce synergistic antimicrobial effects on Aspergillus fumigatus, and dissect possible molecular mechanisms of action. Plate inoculation assays combined with drug susceptibility test and cytotoxicity test showed that the combination of AgNPs and domiphen has synergistic antimicrobial effects on A. fumigatus with low cytotoxicity. Gene Ontology (GO) enrichment analysis showed that AgNPs and domiphen inhibit the growth of A. fumigatus by suppressing nitrate assimilation, and purine nucleobase metabolic process and amino acid transmembrane transport, respectively. When the two drugs are combined, AgNPs has epistatic effects on domiphen. Moreover, the combination of AgNPs and domiphen primarily influence secondary metabolites biosynthesis, steroid biosynthesis and nucleotide sugar metabolism of A. fumigatus via Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, protein-protein interactions (PPI) analysis combined with validation experiments showed that the combination of AgNPs and domiphen could enhance the expression of copper transporter and inhibit nitrogen source metabolism. In addition, the synergistic antimicrobial effects could be enhanced or eliminated depending on exogenous addition of copper and nitrogen source, respectively. Taken together, the results of this study provide a theoretical basis and a new strategy for the treatment of IA.

The Lipid Raft-Associated Protein Stomatin Is Required for Accumulation of Dectin-1 in the Phagosomal Membrane and for Full Activity of Macrophages against Aspergillus fumigatus.

Alveolar macrophages belong to the first line of defense against inhaled conidia of the human-pathogenic fungus Aspergillus fumigatus. In lung alveoli, they contribute to phagocytosis and elimination of conidia. As a counterdefense, conidia have a gray-green pigment that enables them to survive in phagosomes of macrophages for some time. Previously, we showed that this conidial pigment interferes with the formation of flotillin-dependent lipid raft microdomains in the phagosomal membrane, thereby preventing the formation of functional phagolysosomes. Besides flotillins, stomatin is a major component of lipid rafts and can be targeted to the membrane. However, only limited information on stomatin is available, in particular on its role in defense against pathogens. To determine the function of this integral membrane protein, a stomatin-deficient macrophage line was generated by CRISPR/Cas9 gene editing. Immunofluorescence microscopy and flow cytometry revealed that stomatin contributes to the phagocytosis of conidia and is important for recruitment of the β-glucan receptor dectin-1 to both the cytoplasmic membrane and phagosomal membrane. In stomatin knockout cells, fusion of phagosomes and lysosomes, recruitment of the vATPase to phagosomes, and tumor necrosis factor alpha (TNF-α) levels were reduced when cells were infected with pigmentless conidia. Thus, our data suggest that stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. IMPORTANCE Stomatin is an integral membrane protein that contributes to the uptake of microbes, e.g., spores of the human-pathogenic fungus Aspergillus fumigatus. By generation of a stomatin-deficient macrophage line by advanced genetic engineering, we found that stomatin is involved in the recruitment of the β-glucan receptor dectin-1 to the phagosomal membrane of macrophages. Furthermore, stomatin is involved in maturation of phagosomes via fostering fusion of phagosomes with lysosomes. The data provide new insights on the important role of stomatin in the immune response against human-pathogenic fungi.