The targeting of stress-response regulators has emerged as a powerful strategy to enhance azole drug efficacy and to abrogate azole drug resistance. Previously, we reported that a damage resistance protein (Dap) family, composed of DapA, DapB, and DapC, could respond to azole stress stimuli in Aspergillus fumigatus, although the exact response mechanisms remain unknown. In this study, RNA-seq analysis found that a total of 180 genes are induced by azole in a dapA-dependent manner. These genes are involved in oxidation-reduction, metabolic processes, and transmembrane transport. Following azole stress stimuli, DapA and DapC consistently show a stable endoplasmic reticulum (ER)-localization pattern. In comparison, the sterol-regulatory element-binding protein SrbA is capable of nuclear translocation from the ER after azole-stress stimuli, suggesting that SrbA, but not Daps, can directly sense azole stress. Moreover, we found that SrbA is required for the normal expression of DapA and DapC but not of DapB. In addition, in the absence of SrbA, the enhanced expression of DapA induced by azole-itraconazole is blocked, indicating that SrbA is required for the DapA response to azole stress. Double mutants together with overexpression experiments suggest that DapA might act downstream of SrbA to respond to azole stress stimuli. Compared with the ΔsrbA strain, no additional increase in sensitivity was observed in the double mutants ΔsrbAΔdapB and ΔsrbAΔdapC, indicating that DapA might be of central importance in the response to azole drugs. Thus, our findings demonstrate that Dap proteins indirectly sense azole stress and link the function of the azole stress-regulator SrbA with the role of Daps in azole susceptibility.

•Afmac1 plays an important role for growth and development under low Cu conditions.•Over-expression of transporters ctrA2 and ctrC can rescue the AfmacA-deletion defects.•ctrA2 and ctrC are expressed in an Afmac1-dependent manner upon Cu starvation.•Ctr proteins coordinately function to adapt to Cu environments.•Afmac1 is required for pathogenesis in vivo.Copper (Cu) is an essential trace element and is regarded as an important virulence factor in fungal pathogens. Previous studies suggest that a putative Cu-sensing transcription factor Mac1 and the Cu transporter Ctr family play important roles during fungal development and virulence. However, how Cu importers of the Ctr family are involved in the Cu acquisition and what is the functional relationship between them have not been fully investigated yet. Here, we demonstrate that the yeast Mac1 homolog in the opportunistic human pathogen Aspergillus fumigatus is required during colony development under low Cu conditions. Transcriptional profiling combined with LacZ reporter analyses indicate that Cu transporters ctrA2 and ctrC are expressed in an Afmac1-dependent manner upon Cu starvation, and over-expression of ctrA2 or ctrC transporters almost completely rescue the Afmac1-deletion defects, suggesting a redundancy of both transporters in Afmac1-mediated Cu uptake. Genetic analysis showed that ctrC may play a dominant role against Cu starvation relative to ctrA2 and elevated expression of ctrA2 can compensate for ctrC deletion under Cu starvation. Interestingly, both ctrA2 and ctrC deletions can suppress ctrB deletion colony defects. Our findings suggest that Ctr family proteins might coordinately regulate their functions to adapt to different Cu environments. Compared to yeast homologs, Cu family proteins in A. fumigatus may have their own working styles. Most importantly, the Afmac1 deletion strain shows a significantly attenuated pathogenicity in the neutropenic immunocompromised (a combination of cyclophosphamide and hydrocortisone) mice model, demonstrating that Afmac1 is required for pathogenesis in vivo.

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.

The growing use of immunosuppressive therapies has resulted in a dramatic increased incidence of invasive fungal infections (IFIs) caused by Aspergillus fumigatus, a common pathogen, and is also associated with a high mortality rate. Azoles are the primary guideline-recommended therapy agents for first-line treatment and prevention of IFIs. However, increased azole usage in medicinal and agricultural settings has caused azole-resistant isolates to repeatedly emerge in the environment, resulting in a significant threat to human health. In this review, we present and summarize current research on the resistance mechanisms of azoles in A. fumigatus as well as efficient susceptibility testing methods. Moreover, we analyze and discuss the putative clinical (bedside) indication of these findings from bench work.

•The excreted polysaccharide of Pleurotus eryngii reduces the lipid accumulation.•EP is consisted of glucose, galactose and mannose with a MW of 36.67 kDa.•EP decreases the lipid uptake by down-regulating the scavenger receptor-CD36.•The culture recipe for EP production was optimized by orthogonal matrix method.Previous study has verified the polysaccharide from the fruiting body of Pleurotus eryngii (PEPE) is capable of decreasing the lipid content in both of cell-line and mouse model. However, little is known about underlying mechanisms and whether this bioactive polysaccharide exists in submerged culture. Here, we verified the excreted polysaccharides EP and EP-1 from submersion culture of P. eryngii have the remarkable inhibitory effects on lipid accumulation in macrophage-derived foam cells. Structure analysis indicates EP-1 consists of d-types of glucose, galactose and mannose with the main β(1 → 3)-glucan glycosidic linkage branched at O-6 by α-d-glucose while EP digested by β-1,3-glucanase fails to decrease the lipid accumulation, suggesting that the special structure is essential for its function. Expression analysis suggests that EP is able to cause the down-regulation of the scavenger receptor-CD36 on both transcription and protein levels. Most importantly, EP can be obtained by fermentation in a mass-production.

•A macrophage-derived foam cell model induced by oxidized low-density lipoprotein.•Different types of edible mushrooms have the diverse polysaccharide components.•P. eryngii contains the bioactive polysaccharides PEPE with 30–38 kDa.•Purified PEPE-A1 and A2 are composed of glucose mannose, and galactose.•P. eryngii polysaccharides have remarkable ability to inhibit lipid accumulation.Mushrooms have a great potential for the production of useful bioactive metabolites. To explore the bioactive compounds from edible mushrooms for interfering with the development of macrophage-derived foam cells, which is recognized as the hallmark of early atherosclerosis, eight types of mushrooms polysaccharides had been selected to be tested. Consequently, different mushrooms polysaccharides displayed diverse component profiles. Of polysaccharides that we tested, the Pleurotus eryngii polysaccharide had the strongest inhibitory effect on lipid accumulation. Furthermore, through fractionation of DEAE-52 and Sephadex G-100, the polysaccharide from P. eryngii had been successfully purified and identified. By the analysis of IR, GC, and HPLC, the purified polysaccharide was estimated to be 30–38 kDa for the average molecular weight with the monosaccharide composition mainly composed of D-types of mannose, glucose and galactose. Findings presented in this report firstly provide direct evidence, which links the purified polysaccharide moiety with the biological function in foam-cell model.

The potential biological functions of Pleurotuseryngii have long been described in Chinese traditional medicine. However, little is known about the bioactive components of P.eryngii and their functions in liver injury and high-fat induced animal models. The present study thoroughly investigated the comprehensive functions of water-soluble polysaccharidic extract of P.eryngii (PEPE) using invivo mouse models and invitro biochemistry tests. PEPE could significantly increase the activities of antioxidant enzymes and effectively remove the free radicals in a liver-injury mouse model. Furthermore, in a high-fat-load mouse model, PEPE not only remarkably decreased the lipid levels of total cholesterol, total triglyceride, and low-density lipoprotein cholesterol, but also resulted in an increase in high-density lipoprotein cholesterol. Histopathological observations indicated that PEPE could effectively prevent excessive lipid formation in liver tissue. Our data suggest that PEPE can be used as a valuable functional food additive for hypolipidemic and hepatoprotective treatments.Highlights► Polysaccharidic extract of Pleurotuseryngii possesses scavenging ability of free radicals. ► PEPE has hepatoprotective function in a liver injury mouse model. ► PEPE acts as a good therapeutic candidate for hyperlipidemia. ► PEPE can be used as a valuable functional food additive.

The importance of proper ion channel trafficking is underpinned by a number of channel-linked genetic diseases whose defect is associated with failure to reach the cell surface. Conceptually, it is reasonable to suggest that the function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane, which is determined jointly by the secretory and endocytic pathways. Yet the precise mechanisms of the entire ion channel trafficking pathway remain unknown. Here, we directly demonstrate that proper membrane localization of a small-conductance Ca2+-activated K+ channel (SK2 or KCa2.2) is dependent on its interacting protein, α-actinin2, a major F-actin crosslinking protein. SK2 channel localization on the cell-surface membrane is dynamically regulated, and one of the critical steps includes the process of cytoskeletal anchoring of SK2 channel by its interacting protein, α-actinin2, as well as endocytic recycling via early endosome back to the cell membrane. Consequently, alteration of these components of SK2 channel recycling results in profound changes in channel surface expression. The importance of our findings may transcend the area of K+ channels, given that similar cytoskeletal interaction and anchoring may be critical for the membrane localization of other ion channels in neurons and other excitable cells.

•McuA is a mitochondrial calcium uniporter homolog.•AgcA is a mitochondrial carrier protein AGC1/MICU1 homolog.•McuA, but not AgcA contributes to mitochondrial Ca2+ uptake.•Loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress.•Ca2+-binding sites in McuA are required for mitochondrial calcium uptake and stress responses.Ca2+ uptake into mitochondria plays a central role in cell physiology by stimulating ATP production, shaping cytosolic Ca2+ transients and regulating cell survival or death. Although this system has been studied extensively in mammalian cells, the physiological implications of Ca2+ uptake into mitochondria in fungal cells are still unknown. In this study, a bi-directional best-hit BLASTP search revealed that the genome of Aspergillus fumigatus encodes a homolog of a putative mitochondrial Ca2+ uniporter (MCU) and a mitochondrial carrier protein AGC1/MICU1 homolog. Both putative homologs are mitochondrially localized and required for the response to azole and oxidative stress such that the loss of either McuA or AgcA results in reduced susceptibility to azole and oxidative stress, suggesting a role in environmental stress adaptation. Overexpressing mcuA restores the azole-resistance phenotype of the ΔagcA strain to wild-type levels, but not vice versa, indicating McuA plays a dominant role during these stress responses. Using a mitochondrially targeted version of the calcium-sensitive photoprotein aequorin, we found that only mcuA deletion leads to dysfunctional [Ca2+]mt and [Ca2+]c homeostasis, suggesting that McuA, but not AgcA, contributes to Ca2+ uptake into mitochondria. Further point-mutation experiments combined with extracellular Ca2+ chelator treatment verified that two predicted Ca2+-binding sites in McuA are required for Ca2+ uptake into mitochondria and stress responses through the regulation of [Ca2+]c homeostasis.

Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca2+. Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP–CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP–CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP–CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.

Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95–100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.

Polarized growth is a central feature in eukaryotes. Establishment and maintenance of cell polarity are coordinated by signaling pathways. In this study, we have identified MobB is required for the regulation of cell polarity in Aspergillus nidulans. Depletion of MobB by alcA (p) promoter repression or deletion of MobB abolished conidiation completely, and induced severe growth defects. mobB mutants showed abnormal nuclear segregation with increased number of nuclei in spores, but the formation of septa occurred among dividing cells. The phenotype of mobB in A. nidulans is similar to that of cotA. Furthermore, we verified that MobB interacted with CotA to function as a complex. Interestingly, both mobB and cotA deletion mutants clearly exhibited filament elongation by using environmental osmotic stress in the media. However, calcium channel blocker or chelator inhibited phenotype suppression of mobB or cotA mutants. These results suggest that Ca2+ is potentially involved in the response to the suppression coupled with osmotic stabilizer. This is the first report of the function of MobB in A. nidulans. We propose that the MobB/CotA complex, a component in the conserved RAM-signaling pathway, serves an important role in cell morphogenesis.

Polarized growth is a central feature in eukaryotes. Establishment and maintenance of cell polarity are coordinated by signaling pathways. In this study, we have identified MobB is required for the regulation of cell polarity in Aspergillus nidulans. Depletion of MobB by alcA (p) promoter repression or deletion of MobB abolished conidiation completely, and induced severe growth defects. mobB mutants showed abnormal nuclear segregation with increased number of nuclei in spores, but the formation of septa occurred among dividing cells. The phenotype of mobB in A. nidulans is similar to that of cotA. Furthermore, we verified that MobB interacted with CotA to function as a complex. Interestingly, both mobB and cotA deletion mutants clearly exhibited filament elongation by using environmental osmotic stress in the media. However, calcium channel blocker or chelator inhibited phenotype suppression of mobB or cotA mutants. These results suggest that Ca2+ is potentially involved in the response to the suppression coupled with osmotic stabilizer. This is the first report of the function of MobB in A. nidulans. We propose that the MobB/CotA complex, a component in the conserved RAM-signaling pathway, serves an important role in cell morphogenesis.

•Azole antifungal activity is affected by extracellular Ca2+ concentration.•Calcium influx response induced by azole antifungals is inhibited by EGTA or BAPTA.•EGTA and FK506 can regulate accumulation of the azole-mimicking substrate Rh123.•Ca2+ responsive genes are up-regulated transiently by azole antifungals.•EGTA can be used as adjunct azole antifungals for aspergillosis in vivo.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 [Ca2+]c increased sharply but the addition of calcium chelator-EGTA or BAPTA almost blocks the calcium influx responses, resulted in the dramatically decreasing of [Ca2+]c transient. Real-time PCR analysis verified that six-tested Ca2+-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.

•AfCpcB encodes a yeast Cpc2 and mammalian RACK1 ortholog.•AfCpcB is a cytoplasmic localized and predicted Gβ-like protein.•AfCpcB is required for hyphal growth, conidiophore morphology and pathogenicity.•AfCpcB has its specific functions compared to the Gα subunit of AfGpaB.•Yeast cpcB orthologs can functionally substitute for AfcpcB in hyphal growth.CpcB (cross pathway control B) encodes a yeast Cpc2 and mammalian RACK1 (receptor for activated protein kinase C) ortholog, which is a WD repeat protein with functional homology to the β subunit of heterotrimeric G proteins in Aspergillus fumigatus. Previous study has reported that CpcB governs growth and development in both A. fumigatus and Aspergillus nidulans. However, little is known about the functional identities of CpcB orthologs and their relationships with G protein complexes. In this study, we verified that cytoplasmic AfCpcB acts as a Gβ-like protein ortholog and plays important roles in hyphal growth, conidiophore morphology, cell wall integrity, and virulence in A. fumigatus. Furthermore, double deletion of AfcpcB and AfgpaB (Gα) causes a similar phenotype to AfgpaB mutant with abnormal multiple septa conidiophores but exhibits sparse conidiation with white and fluffy colonies. Thus, the exacerbated conidiation defect suggests that AfcpcB has its own specific function compared to the Gα subunit of AfgpaB or the G-protein complex. In addition, complementation assays using AfcpcB orthologs of A. nidulans and yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans) suggest that all tested fungal AfcpcB orthologs under the A. fumigatus native promoter can largely restore hyphal growth defects in AfcpcB deletion mutant, but only the A. nidulans cpcB ortholog completely rescues the ΔAfcpcB conidiation defect, suggesting that CpcB acts as a Gβ-like protein ortholog in the Aspergilli, but may have unique and important unexplored functions that required for conidiation, which is absent in yeast.