Structural adaptation of fungal cell wall in hypersaline environment.

Halophilic fungi thrive in hypersaline habitats and face a range of extreme conditions. These fungal species have gained considerable attention due to their potential applications in harsh industrial processes, such as bioremediation and fermentation under unfavorable conditions of hypersalinity, low water activity, and extreme pH. However, the role of the cell wall in surviving these environmental conditions remains unclear. Here we employ solid-state NMR spectroscopy to compare the cell wall architecture of Aspergillus sydowii across salinity gradients. Analyses of intact cells reveal that A. sydowii cell walls contain a rigid core comprising chitin, ß-glucan, and chitosan, shielded by a surface shell composed of galactomannan and galactosaminogalactan. When exposed to hypersaline conditions, A. sydowii enhances chitin biosynthesis and incorporates α-glucan to create thick, stiff, and hydrophobic cell walls. Such structural rearrangements enable the fungus to adapt to both hypersaline and salt-deprived conditions, providing a robust mechanism for withstanding external stress. These molecular principles can aid in the optimization of halophilic strains for biotechnology applications.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Cell wall of Aspergillus fumigatus: Variability and response to stress.

The fungal cell is surrounded by a thick cell wall which obviously play an essential role in the protection of the fungus against external aggressive environments. In spite of 50 years of studies, the cell wall remains poorly known and especially its constant modifications during growth as well as environmental changes is not well appreciated. This review focus on the cell wall changes seen between different fungal stages and cell populations with a specific view to explain the resistance to stresses.

Conidium Specific Polysaccharides in Aspergillus fumigatus.

Earlier studies have shown that the outer layers of the conidial and mycelial cell walls of Aspergillus fumigatus are different. In this work, we analyzed the polysaccharidome of the resting conidial cell wall and observed major differences within the mycelium cell wall. Mainly, the conidia cell wall was characterized by (i) a smaller amount of α-(1,3)-glucan and chitin; (ii) a larger amount of ß-(1,3)-glucan, which was divided into alkali-insoluble and water-soluble fractions, and (iii) the existence of a specific mannan with side chains containing galactopyranose, glucose, and N-acetylglucosamine residues. An analysis of A. fumigatus cell wall gene mutants suggested that members of the fungal GH-72 transglycosylase family play a crucial role in the conidia cell wall ß-(1,3)-glucan organization and that α-(1,6)-mannosyltransferases of GT-32 and GT-62 families are essential to the polymerization of the conidium-associated cell wall mannan. This specific mannan and the well-known galactomannan follow two independent biosynthetic pathways.

The New GPI-Anchored Protein, SwgA, Is Involved in Nitrogen Metabolism in the Pathogenic Filamentous Fungus Aspergillus fumigatus.

GPI-anchored proteins display very diverse biological (biochemical and immunological) functions. An in silico analysis has revealed that the genome of Aspergillus fumigatus contains 86 genes coding for putative GPI-anchored proteins (GPI-APs). Past research has demonstrated the involvement of GPI-APs in cell wall remodeling, virulence, and adhesion. We analyzed a new GPI-anchored protein called SwgA. We showed that this protein is mainly present in the Clavati of Aspergillus and is absent from yeasts and other molds. The protein, localized in the membrane of A. fumigatus, is involved in germination, growth, and morphogenesis, and is associated with nitrogen metabolism and thermosensitivity. swgA is controlled by the nitrogen regulator AreA. This current study indicates that GPI-APs have more general functions in fungal metabolism than cell wall biosynthesis.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Study on the mechanisms of action of berberine combined with fluconazole against fluconazole-resistant strains of Talaromyces marneffei.

Talaromyces (Penicillium) marneffei (T. marneffei) is a thermally dimorphic fungus that can cause opportunistic systemic mycoses. Our previous study demonstrated that concomitant use of berberine (BBR) and fluconazole (FLC) showed a synergistic action against FLC-resistant T. marneffei (B4) in vitro. In this paper, we tried to figure out the antifungal mechanisms of BBR and FLC in T. marneffei FLC-resistant. In the microdilution test, the minimum inhibitory concentration (MIC) of FLC was 256 µg/ml before FLC and BBR combination, and was 8 µg/ml after combination, the partial inhibitory concentration index (FICI) of B4 was 0.28. After the treatments of BBR and FLC, the studies revealed that (i) increase reactive oxygen species (ROS), (ii) reduce ergosterol content, (iii) destroy the integrity of cell wall and membrane, (iv) decrease the expression of genes AtrF, MDR1, PMFCZ, and Cyp51B however ABC1 and MFS change are not obvious. These results confirmed that BBR has antifungal effect on T. marneffei, and the combination with FLC can restore the susceptibility of FLC-resistant strains to FLC, and the reduction of ergosterol content and the down-regulation of gene expression of AtrF, Mdr1, PMFCZ, and Cyp51B are the mechanisms of the antifungal effect after the combination, which provides a theoretical basis for the application of BBR in the treatment of Talaromycosis and opens up new ideas for treatment of Talaromycosis.

Central Sugar Metabolism and the Cell Wall.

The human opportunistic pathogen Aspergillus fumigatus is recognized for its versatile cell wall when it comes to remodeling its components in adaptation to external threats, and this remodeling renders it refractory to antifungals targeting cell wall biosynthesis. A specific role for general sugar metabolism in the regulation of the synthesis of cell wall polymers has been previously demonstrated. Delving deeper into central sugar metabolism may reveal unexpected fundamental aspects in cell wall construction, as shown by the work of Zhou and coworkers (Y. Zhou, K. Yan, Q. Qin, O.G. Raimi, et al., mBio 13:e01426-22, 2022, https://doi.org/10.1128/mbio.01426-22) on the roles of the phosphoglucose isomerase of A. fumigatus in cell wall biosynthesis.

Modern Biophysics Redefines Our Understanding of Fungal Cell Wall Structure, Complexity, and Dynamics.

Even though the cell wall has been recognized as a crucial protective organelle for fungi, essential for its virulence and a unique emblem of this kingdom, biosynthesis of this organelle remains poorly understood. Our knowledge was based mainly in the past on the chemical analysis of cell wall mutants and on the biochemical study of a few synthases and transglycosidases. Recent developments in biophysical equipment and methods, such as solid-state nuclear magnetic resonance or cryo-electron microscopy, have promoted a better appreciation of the spatiotemporal dynamics of cell wall biosynthesis. The new information will be presented here with the cell wall of the human opportunistic pathogen Aspergillus fumigatus.

Comparative Genomics Reveals a Single Nucleotide Deletion in pksP That Results in White-Spore Phenotype in Natural Variants of Aspergillus fumigatus.

Aspergillus fumigatus is a potentially deadly opportunistic human pathogen. A. fumigatus has evolved a variety of mechanisms to evade detection by the immune system. For example, the conidium surface is covered in a layer of 1,8-dihydroxynaphthalene (DHN) melanin which masks the antigen macrophages use for recognition. DHN melanin also protects conidia from ultraviolet radiation and gives A. fumigatus conidia their characteristic green-grayish color. Here, we conducted genomic analysis of two closely related white-spore natural variants of A. fumigatus in comparison to two closely related green-spore isolates to identify a genetic basis of the white-spore phenotype. Illumina whole-genome resequencing data of the four isolates was used to identify variants that were shared in the white-spore isolates and different from both the green-spore isolates and the Af293 reference genome (which is also a green-spore isolate). We identified 4,279 single nucleotide variants and 1,785 insertion/deletions fitting this pattern. Among these, we identified 64 variants predicted to be high impact, loss-of-function mutations. One of these variants is a single nucleotide deletion that results in a frameshift in pksP (Afu2g17600), the core biosynthetic gene in the DHN melanin encoding gene cluster. The frameshift mutation in the white-spore isolates leads to a truncated protein in which a phosphopantetheine attachment site (PP-binding domain) is interrupted and an additional PP-binding domain and a thioesterase domain are omitted. Growth rate analysis of white-spore and green-spore isolates at 37°C and 48°C revealed that white-spore isolates are thermosensitive. Growth rate of A. fumigatus Af293 and a pksP null mutant in the Af293 background suggests pksP is not directly involved in the thermosensitivity phenotype. Further, our study identified a mutation in a gene (Afu4g04740) associated with thermal sensitivity in yeasts which could also be responsible for the thermosensitivity of the white-spore mutants. Overall, we used comparative genomics to identify the mutation and protein alterations responsible for the white-spore phenotype of environmental isolates of A. fumigatus.

A molecular vision of fungal cell wall organization by functional genomics and solid-state NMR.

Vast efforts have been devoted to the development of antifungal drugs targeting the cell wall, but the supramolecular architecture of this carbohydrate-rich composite remains insufficiently understood. Here we compare the cell wall structure of a fungal pathogen Aspergillus fumigatus and four mutants depleted of major structural polysaccharides. High-resolution solid-state NMR spectroscopy of intact cells reveals a rigid core formed by chitin, ß-1,3-glucan, and α-1,3-glucan, with galactosaminogalactan and galactomannan present in the mobile phase. Gene deletion reshuffles the composition and spatial organization of polysaccharides, with significant changes in their dynamics and water accessibility. The distribution of α-1,3-glucan in chemically isolated and dynamically distinct domains supports its functional diversity. Identification of valines in the alkali-insoluble carbohydrate core suggests a putative function in stabilizing macromolecular complexes. We propose a revised model of cell wall architecture which will improve our understanding of the structural response of fungal pathogens to stresses.

Structural Polymorphism of Chitin and Chitosan in Fungal Cell Walls From Solid-State NMR and Principal Component Analysis.

Chitin is a major carbohydrate component of the fungal cell wall and a promising target for novel antifungal agents. However, it is technically challenging to characterize the structure of this polymer in native cell walls. Here, we recorded and compared 13C chemical shifts of chitin using isotopically enriched cells of six Aspergillus, Rhizopus, and Candida strains, with data interpretation assisted by principal component analysis (PCA) and linear discriminant analysis (LDA) methods. The structure of chitin is found to be intrinsically heterogeneous, with peak multiplicity detected in each sample and distinct fingerprints observed across fungal species. Fungal chitin exhibits partial similarity to the model structures of α- and γ-allomorphs; therefore, chitin structure is not significantly affected by interactions with other cell wall components. Addition of antifungal drugs and salts did not significantly perturb the chemical shifts, revealing the structural resistance of chitin to external stress. In addition, the structure of the deacetylated form, chitosan, was found to resemble a relaxed two-fold helix conformation. This study provides high-resolution information on the structure of chitin and chitosan in their cellular contexts. The method is applicable to the analysis of other complex carbohydrates and polymer composites.

Uncoupling of IL-6 signaling and LC3-associated phagocytosis drives immunoparalysis during sepsis.

Immune deactivation of phagocytes is a central event in the pathogenesis of sepsis. Herein, we identify a master regulatory role of IL-6 signaling on LC3-associated phagocytosis (LAP) and reveal that uncoupling of these two processes during sepsis induces immunoparalysis in monocytes/macrophages. In particular, we demonstrate that activation of LAP by the human fungal pathogen Aspergillus fumigatus depends on ERK1/2-mediated phosphorylation of p47phox subunit of NADPH oxidase. Physiologically, autocrine IL-6/JAK2/Ninein axis orchestrates microtubule organization and dynamics regulating ERK recruitment to the phagosome and LC3+ phagosome (LAPosome) formation. In sepsis, loss of IL-6 signaling specifically abrogates microtubule-mediated trafficking of ERK, leading to defective activation of LAP and impaired killing of bacterial and fungal pathogens by monocytes/macrophages, which can be selectively restored by IL-6 supplementation. Our work uncovers a molecular pathway linking IL-6 signaling with LAP and provides insight into the mechanisms underlying immunoparalysis in sepsis.

Functional Genomic and Biochemical Analysis Reveals Pleiotropic Effect of Congo Red on Aspergillus fumigatus.

Inhibition of fungal growth by Congo red (CR) has been putatively associated with specific binding to ß-1,3-glucans, which blocks cell wall polysaccharide synthesis. In this study, we searched for transcription factors (TFs) that regulate the response to CR and interrogated their regulon. During the investigation of the susceptibility to CR of the TF mutant library, several CR-resistant and -hypersensitive mutants were discovered and further studied. Abnormal distorted swollen conidia called Quasimodo cells were seen in the presence of CR. Quasimodo cells in the resistant mutants were larger than the ones in the sensitive and parental strains; consequently, the conidia of the resistant mutants absorbed more CR than the germinating conidia of the sensitive or parental strains. Accordingly, this higher absorption rate by Quasimodo cells resulted in the removal of CR from the culture medium, allowing a subset of conidia to germinate and grow. In contrast, all resting conidia of the sensitive mutants and the parental strain were killed. This result indicated that the heterogeneity of the conidial population is essential to promote the survival of Aspergillus fumigatus in the presence of CR. Moreover, amorphous surface cell wall polysaccharides such as galactosaminogalactan control the influx of CR inside the cells and, accordingly, resistance to the drug. Finally, long-term incubation with CR led to the discovery of a new CR-induced growth effect, called drug-induced growth stimulation (DIGS), since the growth of one of them could be stimulated after recovery from CR stress.IMPORTANCE The compound Congo red (CR) has been historically used for coloring treatment and histological examination as well to inhibit the growth of yeast and filamentous fungi. It has been thought that CR binds to ß-1,3-glucans in the fungal cell wall, disrupting the organization of the cell wall structure. However, other processes have been implicated in affecting CR sensitivity. Here, we explore CR susceptibility through screening a library of genetic null mutants. We find several previously uncharacterized genetic regulators important for CR susceptibility. Through biochemical and molecular characterization, we find cell membrane permeability to be important. Additionally, we characterize a novel cell type, Quasimodo cells, that occurs upon CR exposure. These cells take up CR, allowing the growth of the remaining fungi. Finally, we find that priming with CR can enhance long-term growth in one mutant.

Data of common and species-specific transcriptional host responses to pathogenic fungi.

Using a comparative RNA-Sequencing based transcriptional profiling approach, responses of primary human peripheral blood mononuclear cells (PBMCs) to common human pathogenic fungi have been characterized (Bruno et al. Computational and Structural Biology Journal). Primary human PBMCs were stimulated in vitro with the fungi A. fumigatus, C. albicans, and R. oryzae after which RNA was isolated and sequenced. From raw sequencing reads differential expressed genes in response to the different fungi where calculated by comparison with unstimulated cells. By overlapping differentially expressed genes in response to the pathogenic fungi A. fumigatus, C. albicans, and R. oryzae a dataset was generated that encompasses a common response to these three distinct fungi as well as species-specific responses. Here we present datasets on these common and species-specific responses that complement the original study (Bruno et al. Computational and Structural Biology Journal). These data serve to facilitate further fundamental research on the immune response to opportunistic pathogenic fungi such as A. fumigatus, C. albicans, and R. oryzae.

Species-Specific Immunological Reactivities Depend on the Cell-Wall Organization of the Two Aspergillus, Aspergillus fumigatus and A. flavus.

Although belong to the same genus, Aspergillus fumigatus is primarily involved in invasive pulmonary infection, whereas Aspergillus flavus is a common cause of superficial infection. In this study, we compared conidia (the infective propagules) of these two Aspergillus species. In immunocompetent mice, intranasal inoculation with conidia of A. flavus resulted in significantly higher inflammatory responses in the lungs compared to mice inoculated with A. fumigatus conidia. In vitro assays revealed that the dormant conidia of A. flavus, unlike A. fumigatus dormant conidia, are immunostimulatory. The conidial surface of A. fumigatus was covered by a rodlet-layer, while that of A. flavus were presented with exposed polysaccharides. A. flavus harbored significantly higher number of proteins in its conidial cell wall compared to A. fumigatus conidia. Notably, ß-1,3-glucan in the A. flavus conidial cell-wall showed significantly higher percentage of branching compared to that of A. fumigatus. The polysaccharides ensemble of A. flavus conidial cell wall stimulated the secretion of proinflammatory cytokines, and conidial cell wall associated proteins specifically stimulated IL-8 secretion from the host immune cells. Furthermore, the two species exhibited different sensitivities to antifungal drugs targeting cell wall polysaccharides, proposing the efficacy of species-specific treatment strategies. Overall, the species-specific organization of the conidial cell wall could be important in establishing infection by the two Aspergillus species.

Genome Sequence of Aspergillus aculeatinus IC_8, Isolated from an Indoor Air Sample of an Urban Housing Complex in Abidjan, Ivory Coast.

Aspergillus aculeatinus is an industrially important species of Aspergillus section Nigri capable of producing bioactive, antibiotic, and antitumor compounds. We sequenced the genome of a strain of A. aculeatinus that was isolated from the interior of a housing complex in Abidjan, Ivory Coast.

Aspergillus fumigatus, One Uninucleate Species with Disparate Offspring.

Establishment of a fungal infection due to Aspergillus fumigatus relies on the efficient germination of the airborne conidia once they penetrate the respiratory tract. However, the features of conidial germination have been poorly explored and understood in this fungal species as well as in other species of filamentous fungi. We show here that the germination of A. fumigatus is asynchronous. If the nutritional environment and extensive gene deletions can modify the germination parameters for A. fumigatus, the asynchrony is maintained in all germinative conditions tested. Even though the causes for this asynchrony of conidial germination remain unknown, asynchrony is essential for the completion of the biological cycle of this filamentous fungus.