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.

Transcription factors SltA and CrzA reversely regulate calcium homeostasis under calcium-limited conditions.

IMPORTANCE: Calcium ions are ubiquitous intracellular signaling molecules for many signaling pathways regulating the fungal response to stress and antifungal drugs. The concentration of intracellular calcium is tightly regulated in its storage, release, and distribution. CrzA is the best-studied transcription factor that regulates this process under sufficient calcium or other external signals. However, CrzA was excluded from nuclei and then lost transcriptional activation under calcium-limited conditions. The regulators in the Ca2+ signaling pathway under calcium-limited conditions remain unclear. Here, we identified SltA as a key regulator in the Ca2+ signaling pathway under calcium-limited conditions, and the underlying mechanisms were further explored in Aspergillus fumigatus. These findings reveal a transcriptional control pathway that precisely regulates calcium homeostasis under calcium-limited conditions.

Transcription factor CreA is involved in the inverse regulation of biofilm formation and asexual development through distinct pathways in Aspergillus fumigatus.

The exopolysaccharide galactosaminogalactan (GAG) contributes to biofilm formation and virulence in the pathogenic fungus Aspergillus fumigatus. Increasing evidence indicates that GAG production is inversely linked with asexual development. However, the mechanisms underlying this regulatory relationship are unclear. In this study, we found that the dysfunction of CreA, a conserved transcription factor involved in carbon catabolite repression in many fungal species, causes abnormal asexual development (conidiation) under liquid-submerged culture conditions specifically in the presence of glucose. The loss of creA decreased GAG production independent of carbon sources. Furthermore, CreA contributed to asexual development and GAG production via distinct pathways. CreA promoted A. fumigatus GAG production by positively regulating GAG biosynthetic genes (uge3 and agd3). CreA suppressed asexual development in glucose liquid-submerged culture conditions via central conidiation genes (brlA, abaA, and wetA) and their upstream activators (flbC and flbD). Restoration of brlA expression to the wild-type level by flbC or flbD deletion abolished the abnormal submerged conidiation in the creA null mutant but did not restore GAG production. The C-terminal region of CreA was crucial for the suppression of asexual development, and the repressive domain contributed to GAG production. Overall, CreA is involved in GAG production and asexual development in an inverse manner.

Filamentous fungal biofilms: Conserved and unique aspects of extracellular matrix composition, mechanisms of drug resistance and regulatory networks in Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is an ubiquitous mold that can cause invasive pulmonary infections in immunocompromised patients. Within the lung, A. fumigatus forms biofilms that can enhance resistance to antifungals and immune defenses, highlighting the importance of defining the mechanisms underlying biofilm development and associated emergent properties. A. fumigatus biofilms display a morphology and architecture that is distinct from bacterial and yeast biofilms. Moreover, A. fumigatus biofilms display unique characteristics in the composition of their extracellular matrix (ECM) and the regulatory networks governing biofilm formation. This review will discuss our current understanding of the form and function of A. fumigatus biofilms, including the unique components of ECM matrix, potential drug resistance mechanisms, the regulatory networks governing A. fumigatus biofilm formation, and potential therapeutics targeting these structures.

Spt20, a Structural Subunit of the SAGA Complex, Regulates Aspergillus fumigatus Biofilm Formation, Asexual Development, and Virulence.

The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. Previous work showed that in A. fumigatus, the Lim domain-binding protein PtaB can form a complex with the sequence-specific transcription factor SomA for regulating GAG biosynthesis, biofilm formation, and asexual development. However, transcriptional coactivators required for biofilm formation in A. fumigatus remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in the GAG biosynthesis, biofilm formation, conidiation, and virulence of A. fumigatus. RNA sequence data demonstrated that Spt20 positively regulates the expression of the GAG biosynthesis genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex (known from yeast) could be immunoprecipitated with Spt20, suggesting that Spt20 acts as a structural subunit of the SAGA complex. Furthermore, distinct modules of SAGA regulate GAG biosynthesis, biofilm formation, and asexual development in A. fumigatus to various degrees. In summary, the novel biofilm regulator Spt20 is reported, which plays a crucial role in the regulation of fungal asexual development, GAG biosynthesis, and virulence in A. fumigatus. These findings expand knowledge on the regulatory circuits of the SAGA complex relevant for the biofilm formation and asexual development of A. fumigatus. IMPORTANCE Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors to transcriptional coactivators (chromatin regulators and the general transcription machinery) and their regulators. Previous research indicated that the sequence-specific complex SomA/PtaB regulates the biofilm formation and asexual development of Aspergillus fumigatus. However, transcriptional coactivators working with sequence-specific transcription factors to regulate A. fumigatus biofilm formation remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a novel regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in galactosaminogalactan (GAG) production, conidiation, and virulence. Moreover, nearly all modules of the SAGA complex were required for the biofilm formation and asexual development of A. fumigatus. These results establish the SAGA complex as a transcriptional coactivator required for the biofilm formation and asexual development of A. fumigatus.

A Transient Receptor Potential-like Calcium Ion Channel in the Filamentous Fungus Aspergillus nidulans.

Transient Receptor Potential (TRP) proteins constitute a superfamily that encodes transmembrane ion channels with highly diverse permeation and gating properties. Filamentous fungi possess putative TRP channel-encoded genes, but their functions remain elusive. Here, we report that a putative TRP-like calcium channel, trpR, in the filamentous fungus Aspergillus nidulans, performs important roles in conidiation and in adapting to cell wall disruption reagents in a high temperature-induced defect-dependent manner, especially under a calcium-limited culture condition. The genetic and functional relationship between TrpR and the previously identified high-affinity calcium channels CchA/MidA indicates that TrpR has an opposite response to CchA/MidA when reacting to cell wall disruption reagents and in regulating calcium transients. However, a considerable addition of calcium can rescue all the defects that occur in TrpR and CchA/MidA, meaning that calcium is able to bypass the necessary requirement. Nevertheless, the colocalization at the membrane of the Golgi for TrpR and the P-type Golgi Ca2+ ATPase PmrA suggests two channels that may work as ion transporters, transferring Ca2+ from the cytosol into the Golgi apparatus and maintaining cellular calcium homeostasis. Therefore, combined with data for the trpR deletion mutant revealing abnormal cell wall structures, TrpR works as a Golgi membrane calcium ion channel that involves cell wall integration.

The Transcription Factor SomA Synchronously Regulates Biofilm Formation and Cell Wall Homeostasis in Aspergillus fumigatus.

Polysaccharides are key components of both the fungal cell wall and biofilm matrix. Despite having distinct assembly and regulation pathways, matrix exopolysaccharide and cell wall polysaccharides share common substrates and intermediates in their biosynthetic pathways. It is not clear, however, if the biosynthetic pathways governing the production of these polysaccharides are cooperatively regulated. Here, we demonstrate that cell wall stress promotes production of the exopolysaccharide galactosaminogalactan (GAG)-depend biofilm formation in the major fungal pathogen of humans Aspergillus fumigatus and that the transcription factor SomA plays a crucial role in mediating this process. A core set of SomA target genes were identified by transcriptome sequencing and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq). We identified a novel SomA-binding site in the promoter regions of GAG biosynthetic genes agd3 and ega3, as well as its regulators medA and stuA Strikingly, this SomA-binding site was also found in the upstream regions of genes encoding the cell wall stress sensors, chitin synthases, and ß-1,3-glucan synthase. Thus, SomA plays a direct regulation of both GAG and cell wall polysaccharide biosynthesis. Consistent with these findings, SomA is required for the maintenance of normal cell wall architecture and compositions in addition to its function in biofilm development. Moreover, SomA was found to globally regulate glucose uptake and utilization, as well as amino sugar and nucleotide sugar metabolism, which provides precursors for polysaccharide synthesis. Collectively, our work provides insight into fungal adaptive mechanisms in response to cell wall stress where biofilm formation and cell wall homeostasis were synchronously regulated.IMPORTANCE The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking ß-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and ß-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

Precise Expression of Afmed15 Is Crucial for Asexual Development, Virulence, and Survival of Aspergillus fumigatus.

The rise of drug resistance in fungal pathogens is becoming a serious problem owing to the limited number of antifungal drugs available. Identifying and targeting factors essential for virulence or development unique to fungal pathogens is one approach to develop novel treatments for fungal infections. In this study, we present the identification and functional characterization of a novel developmental regulator in Aspergillus fumigatus, AfMed15, which contained a conserved Med15_fungal domain, as determined by screening of a mutant library that contained more than 2,000 hygromycin-resistant A. fumigatus transformants. Downregulating the expression of Afmed15 abolished the conidiation and decreased the fungal virulence in an insect model. Strikingly, the overexpression of Afmed15 caused fungal death accompanied by intensive autophagy. RNA sequencing of an Afmed15 overexpression strain revealed that altered gene expression patterns were associated with carbon metabolism, energy metabolism, and translation. Interestingly, the addition of metal ions could partially rescue fungal death caused by the overexpression of Afmed15, indicating that disordered ion homeostasis is a potential reason for the fungal death caused by the overexpression of Afmed15 Considering that the precise expression of Afmed15 is crucial for fungal development, virulence, and survival and that no ortholog was found in humans, Afmed15 is an ideal target for antifungal-drug development.IMPORTANCE The identification and characterization of regulators essential for virulence or development constitute one approach for antifungal drug development. In this study, we screened and functionally characterized Afmed15, a novel developmental regulator in A. fumigatus We demonstrate that the precise transcriptional expression of Afmed15 is crucial for fungal asexual development, virulence, and survival. Downregulating the expression of Afmed15 abolished the conidiation and decreased the fungal virulence in an insect model. In contrast, the overexpression of Afmed15 caused fungal death accompanied by intensive autophagy. Our study provides a foundation for further studies to identify compounds perturbing the expression of Afmed15 that may be used for the prevention of invasive A. fumigatus infections.

The mitochondrial thiamine pyrophosphate transporter TptA promotes adaptation to low iron conditions and virulence in fungal pathogen Aspergillus fumigatus.

Aspergillus fumigatus is the most prevalent airborne fungal pathogen that causes invasive fungal infections in immunosuppressed individuals. Adaptation to iron limited conditions is crucial for A. fumigatus virulence. To identify novel genes that play roles in adaptation to low iron conditions we performed an insertional mutagenesis screen in A. fumigatus. Using this approach, we identified the tptA gene in A. fumigatus, which shares homology with the Saccharomyces cerevisiae thiamine pyrophosphate (ThPP) transporter encoding gene tpc1. Heterologous expression of tpc1 in the tptA deletion mutant completely restored the ThPP auxotrophy phenotype, suggesting that Tpc1 and TptA are functional orthologues. Importantly, TptA was required for adaptation to low iron conditions in A. fumigatus. The ΔtptA mutant had decreased resistance to the iron chelator bathophenanthroline disulfonate (BPS) with severe growth defects. Moreover, loss of tptA decreased the expression of hapX, which is a major transcription factor indispensable for adaptation to iron starvation in A. fumigatus. Overexpression of hapX in the ΔtptA strain greatly rescued the growth defect and siderophore production by A. fumigatus in iron-depleted conditions. Mutagenesis experiments demonstrated that the conserved residues related to ThPP uptake in TptA were also required for low iron adaptation. Furthermore, TptA-mediated adaptation to low iron conditions was found to be dependent on carbon sources. Finally, loss of tptA resulted in the attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrated that the mitochondrial ThPP transporter TptA promotes low iron adaptation and virulence in A. fumigatus.

The Zn2Cys6-type transcription factor LeuB cross-links regulation of leucine biosynthesis and iron acquisition in Aspergillus fumigatus.

Both branched-chain amino acids (BCAA) and iron are essential nutrients for eukaryotic cells. Previously, the Zn2Cys6-type transcription factor Leu3/LeuB was shown to play a crucial role in regulation of BCAA biosynthesis and nitrogen metabolism in Saccharomyces cerevisiae and Aspergillus nidulans. In this study, we found that the A. fumigatus homolog LeuB is involved in regulation of not only BCAA biosynthesis and nitrogen metabolism but also iron acquisition including siderophore metabolism. Lack of LeuB caused a growth defect, which was cured by supplementation with leucine or iron. Moreover, simultaneous inactivation of LeuB and HapX, a bZIP transcription factor required for adaptation to iron starvation, significantly aggravated the growth defect caused by inactivation of one of these regulators during iron starvation. In agreement with a direct role in regulation of both BCAA and iron metabolism, LeuB was found to bind to phylogenetically conserved motifs in promoters of genes involved in BCAA biosynthesis, nitrogen metabolism, and iron acquisition in vitro and in vivo, and was required for full activation of their expression. Lack of LeuB also caused activation of protease activity and autophagy via leucine depletion. Moreover, LeuB inactivation resulted in virulence attenuation of A. fumigatus in Galleria mellonella. Taken together, this study identified a previously uncharacterized direct cross-regulation of BCCA biosynthesis, nitrogen metabolism and iron homeostasis as well as proteolysis.

The Claudin Family Protein FigA Mediates Ca2+ Homeostasis in Response to Extracellular Stimuli in Aspergillus nidulans and Aspergillus fumigatus.

The claudin family protein Fig1 is a unique fungal protein that is involved in pheromone-induced calcium influx and membrane fusion during the mating of Saccharomyces cerevisiae and Candida albicans. Whether and how Fig1 regulates Ca2+ homeostasis in response to extracellular stimuli is poorly understood. Previously, we found Aspergillus nidulans FigA, a homolog of Fig1 in S. cerevisiae, similar to the high-affinity calcium uptake system, is required for normal growth under low-Ca2+ minimal medium. In this study, using the calcium-sensitive photoprotein aequorin to monitor cytosolic free calcium concentration ([Ca2+]c) in living cells, we found that the FigA dysfunction decreases the transient [Ca2+]c induced by a high extracellular calcium stress. Furthermore, FigA acts synergistically with CchA (a high-affinity Ca2+ channel) to coordinate cytoplasmic Ca2+ influx in response to an extracellular Ca2+ stimulus. Moreover, FigA mediates ER stress-induced transient [Ca2+]c in the presence or absence of extracellular calcium. Most importantly, these [Ca2+]c responses mediated by FigA are closely related to its conserved claudin superfamily motif, which is also required for hyphal growth and asexual development in A. nidulans. Finally, the function of FigA in Aspergillus fumigatus, the most common airborne human fungal pathogen was studied. The result showed that the two FigA homologous in A. nidulans and A. fumigatus have a large degree of functional homology not only in asexual development but also in regulating transient [Ca2+]c. Our study expands the knowledge of claudin family protein FigA in Ca2+ homeostasis in response to extracellular stimuli.

Lipid-Lowering Effect of the Pleurotus eryngii (King Oyster Mushroom) Polysaccharide from Solid-State Fermentation on Both Macrophage-Derived Foam Cells and Zebrafish Models.

Hyperlipidemia is a key risk factor in inducing fatty liver, hypertension, atherosclerosis and cerebrovascular diseases. Previous studies have verified that polysaccharides from fruiting bodies (PEPE) of Pleurotus eryngii (king oyster mushroom) are capable of decreasing the lipid content. In this study, the P. eryngii polysaccharide is obtained by solid-state fermentation (PESF) using lignocellulosic wastes, corn-cobs and wheat bran. The high-performance liquid chromatography (HPLC) assays indicate that PESF has a similar composition to that of PEPE. Meanwhile, PESF has no detectable toxicity and is able to significantly inhibit foam-cell formation in murine macrophage cells (RAW264.7) induced by oxidized low-density lipoprotein. Further verification indicates that PESF has lipid-lowering effects during the lipid absorption phase in a zebrafish hyperlipidemia model. Our findings suggest that the P. eryngii polysaccharide from solid-state fermentation (PESF) can be used as a valuable lipid-lowering food additive or raw materials for producing lipid-lowering drugs.

PtaB, a lim-domain binding protein in Aspergillus fumigatus regulates biofilm formation and conidiation through distinct pathways.

The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. The developmental modifiers MedA, StuA, and SomA regulate GAG biosynthesis, but the mechanisms underlying this regulation are poorly understood. PtaB is a lim-domain binding protein that interacts with the transcription factor SomA and is required for normal conidiation and biofilm formation. Disruption of ptaB resulted in impaired GAG production and conidiation in association with a markedly reduced expression of GAG biosynthetic genes (uge3 and agd3), developmental regulators (medA and stuA), and genes involved in the core conidiation pathway. Overexpression of medA and dual overexpression of uge3 and agd3 in the ΔptaB mutant increased biofilm formation but not conidiation, whereas overexpression of core conidiation genes rescued conidiation but not biofilm formation. Overexpression of stuA modestly increased both conidiation and biofilm formation. Analysis of ptaB truncation mutants revealed that overexpression of the lim-domain binding region restored conidiation but not biofilm formation, suggesting that ptaB may govern these processes by interacting with different partners. These studies establish that PtaB governs GAG biosynthesis at the level of substrate availability and polymer deacetylation and that PtaB-mediated biofilm formation and conidiation are largely independent pathways.

The Lectin Chaperone Calnexin Is Involved in the Endoplasmic Reticulum Stress Response by Regulating Ca2+ Homeostasis in Aspergillus nidulans.

The Ca2+-mediated signaling pathway is crucial for environmental adaptation in fungi. Here we show that calnexin, a molecular chaperone located in the endoplasmic reticulum (ER), plays an important role in regulating the cytosolic free calcium concentration ([Ca2+]c) in Aspergillus nidulans Inactivation of calnexin (ClxA) in A. nidulans caused severe defects in hyphal growth and conidiation under ER stress caused by the ER stress-inducing agent dithiothreitol (DTT) or high temperature. Importantly, defects in the ΔclxA mutant were restored by the addition of extracellular calcium. Furthermore, the CchA/MidA complex (the high-affinity Ca2+ channels), calcineurin (calcium/calmodulin-dependent protein phosphatase), and PmrA (secretory pathway Ca2+ ATPase) were required for extracellular calcium-based restoration of the DTT/thermal stress sensitivity in the ΔclxA mutant. Interestingly, the ΔclxA mutant exhibited markedly reduced conidium formation and hyphal growth defects under the low-calcium condition, which is similar to defects caused by mutations in MidA/CchA. Moreover, the phenotypic defects were further exacerbated in the ΔclxA ΔmidA ΔcchA mutant, which suggested that ClxA and MidA/CchA are both required under the calcium-limiting condition. Using the calcium-sensitive photoprotein aequorin to monitor [Ca2+]c in living cells, we found that ClxA and MidA/CchA complex synergistically coordinate transient increase in [Ca2+]c in response to extracellular calcium. Moreover, ClxA, in particular its luminal domain, plays a role in mediating the transient [Ca2+]c in response to DTT-induced ER stress in the absence of extracellular calcium, indicating ClxA may mediate calcium release from internal calcium stores. Our findings provide new insights into the role of calnexin in the regulation of calcium-mediated response in fungal ER stress adaptation.IMPORTANCE Calnexin is a well-known molecular chaperone conserved from yeast to humans. Although it contains calcium binding domains, little is known about the role of calnexin in Ca2+ regulation. In this study, we demonstrate that calnexin (ClxA) in the filamentous fungus Aspergillus nidulans, similar to the high-affinity calcium uptake system (HACS), is required for normal growth and conidiation under the calcium-limiting condition. The ClxA dysfunction decreases the transient cytosolic free calcium concentration ([Ca2+]c) induced by a high extracellular calcium or DTT-induced ER stress. Our findings provide the direct evidence that calnexin plays important roles in regulating Ca2+ homeostasis in addition to its role as a molecular chaperone in fungi. These results provide new insights into the roles of calnexin and expand knowledge of fungal stress adaptation.

A Putative Mitochondrial Iron Transporter MrsA in Aspergillus fumigatus Plays Important Roles in Azole-, Oxidative Stress Responses and Virulence.

Iron is an essential nutrient and enzyme co-factor required for a wide range of cellular processes, especially for the function of mitochondria. For the opportunistic fungal pathogen Aspergillus fumigatus, the ability to obtain iron is required for growth and virulence during the infection process. However, knowledge of how mitochondria are involved in iron regulation is still limited. Here, we show that a mitochondrial iron transporter, MrsA, a homolog of yeast Mrs4p, is critical for adaptation to iron-limited or iron-excess conditions in A. fumigatus. Deletion of mrsA leads to disruption of iron homeostasis with a decreased sreA expression, resulted in activated reductive iron assimilation (RIA) and siderophore-mediated iron acquisition (SIA). Furthermore, deletion of mrsA induces hypersusceptibility to azole and oxidative stresses. An assay for cellular ROS content in ΔmrsA combined with rescue from the mrsA-defective phenotype by the antioxidant reagent L-ascorbic acid indicates that the increased sensitivity of ΔmrsA to the azole itraconazole and to oxidative stress is mainly the result of abnormal ROS accumulation. Moreover, site-directed mutation experiments verified that three conserved histidine residues related to iron transport in MrsA are required for responses to oxidative and azole stresses. Importantly, ΔmrsA causes significant attenuation of virulence in an immunocompromised murine model of aspergillosis. Collectively, our results show that the putative mitochondrial iron transporter MrsA plays important roles in azole- and oxidative-stress responses and virulence by regulating the balance of cellular iron in A. fumigatus.

Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus.

Finely tuned changes in cytosolic free calcium ([Ca2+]c) mediate numerous intracellular functions resulting in the activation or inactivation of a series of target proteins. Palmitoylation is a reversible post-translational modification involved in membrane protein trafficking between membranes and in their functional modulation. However, studies on the relationship between palmitoylation and calcium signaling have been limited. Here, we demonstrate that the yeast palmitoyl transferase ScAkr1p homolog, AkrA in Aspergillus nidulans, regulates [Ca2+]c homeostasis. Deletion of akrA showed marked defects in hyphal growth and conidiation under low calcium conditions which were similar to the effects of deleting components of the high-affinity calcium uptake system (HACS). The [Ca2+]c dynamics in living cells expressing the calcium reporter aequorin in different akrA mutant backgrounds were defective in their [Ca2+]c responses to high extracellular Ca2+ stress or drugs that cause ER or plasma membrane stress. All of these effects on the [Ca2+]c responses mediated by AkrA were closely associated with the cysteine residue of the AkrA DHHC motif, which is required for palmitoylation by AkrA. Using the acyl-biotin exchange chemistry assay combined with proteomic mass spectrometry, we identified protein substrates palmitoylated by AkrA including two new putative P-type ATPases (Pmc1 and Spf1 homologs), a putative proton V-type proton ATPase (Vma5 homolog) and three putative proteins in A. nidulans, the transcripts of which have previously been shown to be induced by extracellular calcium stress in a CrzA-dependent manner. Thus, our findings provide strong evidence that the AkrA protein regulates [Ca2+]c homeostasis by palmitoylating these protein candidates and give new insights the role of palmitoylation in the regulation of calcium-mediated responses to extracellular, ER or plasma membrane stress.

Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans.

The eukaryotic calcium/calmodulin-dependent protein phosphatase calcineurin is crucial for the environmental adaption of fungi. However, the mechanism of coordinate regulation of the response to salt stress by calcineurin and the high-affinity calcium channel CchA in fungi is not well understood. Here we show that the deletion of cchA suppresses the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans Additionally, the hypersensitivity of the ΔcnaA strain to extracellular calcium and cell-wall-damaging agents can be suppressed by cchA deletion. Using the calcium-sensitive photoprotein aequorin to monitor the cytoplasmic Ca(2+) concentration ([Ca(2+)]c) in living cells, we found that calcineurin negatively regulates CchA on calcium uptake in response to external calcium in normally cultured cells. However, in salt-stress-pretreated cells, loss of either cnaA or cchA significantly decreased the [Ca(2+)]c, but a deficiency in both cnaA and cchA switches the [Ca(2+)]c to the reference strain level, indicating that calcineurin and CchA synergistically coordinate calcium influx under salt stress. Moreover, real-time PCR results showed that the dysfunction of cchA in the ΔcnaA strain dramatically restored the expression of enaA (a major determinant for sodium detoxification), which was abolished in the ΔcnaA strain under salt stress. These results suggest that double deficiencies of cnaA and cchA could bypass the requirement of calcineurin to induce enaA expression under salt stress. Finally, YvcA, a member of the transient receptor potential channel (TRPC) protein family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption.IMPORTANCE The feedback inhibition relationship between calcineurin and the calcium channel Cch1/Mid1 has been well recognized from yeast. Interestingly, our previous study (S. Wang et al., PLoS One 7:e46564, 2012, http://dx.doi.org/10.1371/journal.pone.0046564) showed that the deletion of cchA could suppress the hyphal growth defects caused by the loss of calcineurin under salt stress in Aspergillus nidulans In this study, our findings suggest that fungi are able to develop a unique mechanism for adapting to environmental salt stress. Compared to cells cultured normally, the NaCl-pretreated cells had a remarkable increase in transient [Ca(2+)]c Furthermore, we show that calcineurin and CchA are required to modulate cellular calcium levels and synergistically coordinate calcium influx under salt stress. Finally, YvcA, a member of of the TRPC family of vacuolar Ca(2+) channels, was proven to compensate for calcineurin-CchA in fungal salt stress adaption. The findings in this study provide insights into the complex regulatory links between calcineurin and CchA to maintain cytoplasmic Ca(2+) homeostasis in response to different environments.

Comparison of antioxidant and antiproliferation activities of polysaccharides from eight species of medicinal mushrooms.

Polysaccharides from mushrooms including Pleurotus eryngii, P. ostreatus, P. nebrodensis, Lentinus edodes, Hypsizygus marmoreus, Flammulina velutipes, Ganoderma lucidum, and Hericium erinaceus were isolated by water extraction and alcohol precipitation. Our results suggest that all tested polysaccharides have the significant antioxidant capacities of scavenging free radicals (1,1-diphenyl-2-picrylhydrazyl and hydroxyl radicals). Among them, the H. erinaceus polysaccharide exhibits the highest 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity, whereas the L. edodes polysaccharide shows the strongest scavenging ability for hydroxyl radicals. Furthermore, using the MCF-7 breast cancer cell line and HeLa cells, all 8 selected polysaccharides are able to inhibit the proliferation of tumor cells, but the strength of inhibition varied depending on the mushroom species and the concentration used. Notably, G. lucidum polysaccharide shows the highest inhibition activity on MCF-7 cells. By comparison, H. erinaceus polysaccharide has the strongest inhibitory effect on HeLa cells. Moreover, high-performance liquid chromatography with a carbohydrate analysis column showed significant differences in polysaccharide components among these mushrooms. Thus our data suggest that the different species of mushrooms have the variable functions because of their own specific polysaccharide components. The 8 mushroom polysaccharides have the potential to be used as valuable functional food additives or sources of therapeutic agents for antioxidant and cancer treatments, especially polysaccharides from H. erinaceus, L. edodes, and G. lucidum.

Calcium signaling mediates antifungal activity of triazole drugs in the Aspergilli.

Azoles are widely applied and largely effective as antifungals; however, the increasing prevalence of clinically resistant isolates has yet to be matched by approaches to improve the efficacy of antimicrobial therapy. In this study, using the model fungus Aspergillus nidulans and one of the most common human pathogen Aspergillus fumigatus as research materials, we present the evidence that calcium signaling is involved in the azole-antifungals-induced stress-response reactions. In normal media, antifungal-itraconazole (ITZ) is able to induce the [Ca(2+)]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca(2+)]c transient. Real-time PCR analysis verified that six-tested Ca(2+)-inducible genes-two calcium channels (cchA/midA), a calmodulin-dependent phosphatase-calcineurin (cnaA), a transcription factor-crzA, and two calcium transporters (pmrA/pmcA)-could be transiently up-regulated by adding ITZ, indicating these components are involved in the azole stress-response reaction. Defect of cnaA or crzA caused more susceptibility to azole antifungals than did single mutants or double deletions of midA and cchA. Notably, EGTA may influence Rh123 accumulation as an azole-mimicking substrate through the process of the drug absorption. In vivo studies of a Galleria mellonella model identified that the calcium chelator works as an adjunct antifungal agent with azoles for invasive aspergillosis. Most importantly, combination of ITZ and EGTA or ITZ with calcium signaling inhibitor-FK506 greatly enhances the ITZ efficacy. Thus, our study provides potential clues that specific inhibitors of calcium signaling could be clinically useful adjuncts to conventional azole antifungals in the Aspergilli.

Riboflavin level manipulates the successive developmental sequences in Aspergillus nidulans.

Auxotrophic markers are useful in fungal genetic analysis. Among the auxotrophic markers, riboB2 is one of the most commonly used markers in many laboratory strains. However, riboB2 mutants in Aspergillus nidulans confer self-sterility and thus are unable to form hybrid cleistothecia by outcross when both parent strains harbor riboB2 auxotrophic marker under the standard protocol. To assess the role of riboflavin during the different developmental stages of A. nidulans, the limited concentrations of riboflavin were monitored. The commonly used dosage of riboflavin (2.5 µg/ml) in the standard medium recipe is enough for hyphal growth and conidiation in the riboflavin auxotrophic riboB2 mutants (enough at 0.02 and 0.5 µg/ml, respectively) in A. nidulans. However, the dosage is not enough to support mature cleistothecium formation. Furthermore, the self-sterile defects in riboB2 mutants on standard medium could be restored by the addition of 25 µg/ml riboflavin, although the required riboflavin concentrations are varied in different genotype strains in A. nidulans. Most importantly, the outcross between riboB2 mutants could also be achieved by the supply of riboflavin in the sexual developmental stage. Our results highlight the potential roles of auxotrophic markers in the development of fungi and improve the efficiency of the genetic analysis in A. nidulans.