The CCAAT-binding complex mediates azole susceptibility of Aspergillus fumigatus by suppressing SrbA expression and cleavage.

In fungal pathogens, the transcription factor SrbA (a sterol regulatory element-binding protein, SREBP) and CBC (CCAAT binding complex) have been reported to regulate azole resistance by competitively binding the TR34 region (34 mer) in the promoter of the drug target gene, erg11A. However, current knowledge about how the SrbA and CBC coordinately mediate erg11A expression remains limited. In this study, we uncovered a novel relationship between HapB (a subunit of CBC) and SrbA in which deletion of hapB significantly prolongs the nuclear retention of SrbA by increasing its expression and cleavage under azole treatment conditions, thereby enhancing Erg11A expression for drug resistance. Furthermore, we verified that loss of HapB significantly induces the expression of the rhomboid protease RbdB, Dsc ubiquitin E3 ligase complex, and signal peptide peptidase SppA, which are required for the cleavage of SrbA, suggesting that HapB acts as a repressor for these genes which contribute to the activation of SrbA by proteolytic cleavage. Together, our study reveals that CBC functions not only to compete with SrbA for binding to erg11A promoter region but also to affect SrbA expression, cleavage, and translocation to nuclei for the function, which ultimately regulate Erg11A expression and azole resistance.

Calcineurin-mediated intracellular organelle calcium homeostasis is required for the survival of fungal pathogens upon extracellular calcium stimuli.

In eukaryotes, calcium not only is an essential mineral nutrient but also serves as an intracellular second messenger that is necessary for many physiological processes. Previous studies showed that the protein phosphatase-calcineurin protects fungi from toxicity caused by the extracellular calcium; however, little is known about how calcineurin mediates the cellular physiology process for this function. In this study, by monitoring intracellular calcium, particularly by tracking vacuolar calcium dynamics in living cells through a novel procedure using modified aequorin, we found that calcineurin dysfunction systematically caused abnormal intracellular calcium homeostasis in cytosol, mitochondria, and vacuole, leading to drastic autophagy, global organelle fragmentation accompanied with the increased expression of cell death-related enzymes, and cell death upon extracellular calcium stimuli. Notably, all detectable defective phenotypes seen with calcineurin mutants can be significantly suppressed by alleviating a cytosolic calcium overload or increasing vacuolar calcium storage capacity, suggesting toxicity of exogenous calcium to calcineurin mutants is tightly associated with abnormal cytosolic calcium accumulation and vacuolar calcium storage capacity deficiency. Our findings provide insights into how the original recognized antifungal drug target-calcineurin regulates intracellular calcium homeostasis for cell survival and may have important implications for antifungal therapy and clinical drug administration.

GlcA-mediated glycerol-3-phosphate synthesis contributes to the oxidation resistance of Aspergillus fumigatus via decreasing the cellular ROS.

Fungi activate corresponding metabolic pathways in response to different carbon sources to adapt to different environments. Previous studies have shown that the glycerol kinase GlcA that phosphorylates glycerol to the intermediate glycerol-3-phosphate (G3P) is required for the growth of Aspergillus fumigatus when glycerol is used as the sole carbon source. The present study identified there were two putative glycerol kinases, GlcA and GlcB, in A. fumigatus but glycerol activated only glcA promoter but not glcB promoter, although both glcA and glcB could encode glycerol kinase. Under normal culture conditions, the absence of glcA caused no detectable colony phenotypes on glucose and other tested carbon sources except glycerol, indicating dissimilation of glucose and these tested carbon sources bypassed requirement of glcA. Notably, the oxidative stress agent H2O2 on the background of glucose medium clearly induced GlcA expression and promoted G3P synthesis. Deletion and overexpression of glcA elicited sensitivity and resistance to oxidative stress agent H2O2, respectively, accompanied by decrease and increase of G3P production. In addition, the sensitivity to oxidative stress in the glcA mutant was probably associated with dysfunction of mitochondria with a decreased mitochondrial membrane potential and an abnormal accumulation of the cellular reactive oxygen species (ROS). Furthermore, overexpressing the glycerol-3-phosphate dehydrogenase GfdA thatcatalyzes the reduction of dihydroxyacetone phosphate (DHAP) to G3P rescued phenotypes of the glcA null mutant to H2O2. Therefore, the present study suggests that GlcA-involved G3P synthesis participates in oxidative stress tolerance of A. fumigatus via regulating the cellular ROS level.

Electron donor cytochrome b5 is required for hyphal tip accumulation of sterol-rich plasma membrane domains and membrane fluidity in Aspergillus fumigatus.

The electron donor cytochrome b5 (CybE/Cyb5) fuels the activity of the ergosterol biosynthesis-related P450 enzymes/P450s by providing electrons to P450s to promote ergosterol biosynthesis. Previous studies reported that lack of Aspergillus fumigatus (A. fumigatus) CybE reduces the proportion of ergosterol in total sterols and induces severe growth defects. However, the molecular characteristics of CybE and the underlying mechanism for CybE maintaining A. fumigatus growth remain poorly understood. Here, we found that CybE locates at the endoplasmic reticulum by its C-terminus with two transmembrane regions. Therefore, lack of the C-terminus of CybE is able to phenocopy a cybE deletion. Notably, cybE deletion reduced the accumulation of the sterol-rich plasma membrane domains (SRDs, the assembly platform of polarity factors/cell end markers and growth machinery) in hyphal tips and decreased membrane fluidity, which correspond to tardiness of hyphal extension and hypersensitivity to low temperature in cybE deletion mutant. Additionally, overexpressing another electron donor-heme-independent P450 reductase (CPR) significantly rescued growth defects and recovered SRD accumulation in deletion of cybE almost to the wild-type level, suggesting CybE maintaining the growth and deposition of SRDs in hyphal tips attributes to its nature as an electron donor. Protein pull-down assays revealed that CybE probably participates in metabolism and transfer of lipids, construction of cytoskeleton and mitochondria-associated energy metabolism to maintain the SRD accumulation in hyphal tips, membrane fluidity and hyphal extension. Findings in this study give a hint that inhibition of CybE may be an effective strategy for resisting the infection of the human pathogen A. fumigatus Importance Investigating the knowledge of the growth regulation in the human opportunistic pathogen A. fumigatus is conducive to design new antifungal approach. The electron donor cytochrome b5 (CybE) plays a crucial role in maintaining the normal growth of A. fumigatus, however, the potential mechanism remains elusive. Herein, we characterized the molecular features of CybE and found the C-terminus with two transmembrane domains are required for its ER localization and functions. In addition, we demonstrated that CprA, an electron donor-heme-independent P450 reductase, provides a reciprocal function for the missing cytochrome b5 protein-CybE in A. fumigatus CybE maintains the normal growth probably via supporting two crucial physiological processes, the SRD accumulation in hyphal tips and membrane fluidity. Therefore, our finding reveals the mechanisms underlying the regulatory effect of CybE on A. fumigatus growth and indicates that inhibition of CybE might be an effective approach for alleviating A. fumigatus infection.

The association between watching football matches and the risk of cardiovascular events: A meta-analysis.

To comprehensively shed light on whether viewing football games is associated with a higher risk of cardiovascular disease (CVD). Electronic databases were searched through 17 May 2018. All studies focusing on the association between viewing football matches and the fatal or non-fatal CVD were identified. Viewing football matches was associated with a higher risk of fatal overall CVD (RR: 1.06, 95%CI: 1.01-1.12) in both men (RR: 1.13, 95%CI: 1.004-1.28) and women (RR: 1.08, 95%CI: 1.01-1.15). Subgroup analysis showed that failure of the team has a higher risk of fatal overall CVD (RR: 1.29, 95%CI: 1.15-1.45). However, lower risk of fatal overall CVD from spectators was observed when team obtained a victory (RR: 0.80, 95%CI: 0.66-0.96). For non-fatal CVD, viewing football matches was associated with a higher risk of non-fatal overall CVD (RR: 1.24, 95%CI: 1.09-1.41) in both men (RR: 1.73, 95%CI: 1.12-2.69) and women (RR: 1.25, 95%CI: 1.08-1.45). Subgroup analysis showed that viewing football matches was associated with a higher risk of non-fatal myocardial infarction (RR: 1.20, 95%CI: 1.04-1.38) in both men and women (RR: 1.51, 95%CI: 0.99-2.28; RR: 1.21, 95%CI: 1.08-1.36, respectively). No significant increase was found in fatal or non-fatal stroke. Viewing football matches was associated with a higher risk of the fatal and non-fatal CVD, especially in male spectators. The victory of team could have a lower risk of fatal CVD. Therefore, precautionary measures should be required for the reduction of healthcare burden in football matches.

Predicted Glycerol 3-Phosphate Dehydrogenase Homologs and the Glycerol Kinase GlcA Coordinately Adapt to Various Carbon Sources and Osmotic Stress in Aspergillus fumigatus.

Glycerol plays an important role in the adaptation of fungi to various microenvironments and stressors, including heat shock, anoxic conditions and osmotic stress. Glycerol 3-phosphate dehydrogenase (G3PDH) is able to catalyze dihydroxyacetone phosphate to glycerol 3-phosphate (G3P), which is subsequently dephosphorylated into glycerol. However, current knowledge about the functions of G3PDH homologs in glycerol biosynthesis in Aspergillus fumigatus is limited. Here, we show that the A. fumigatus G3PDH gene, gfdA, is crucial for normal colony growth in glucose media under both normoxic and hypoxic conditions. In addition, failure of the overexpression of the gfdA homolog, gfdB, to rescue the phenotype of a gfdA null mutant suggests that gfdA plays a predominant role in the synthesis of G3P and glycerol. However, in a wild-type background, overexpressing either gfdA or gfdB is able to significantly enhance biomass production of mycelia, suggesting that gfdA and gfdB have similar functions in promoting the use of glucose. Interestingly, overexpression of the gene encoding the predicted glycerol kinase, GlcA, which is capable of phosphorylating glycerol to form G3P, significantly rescues the growth defects of gfdA null mutants in glucose media, indicating that the growth defects of gfdA null mutants might be due to the absence of G3P rather than glycerol. Moreover, Western blotting analysis revealed that gfdA is inducibly expressed by osmotic mediators. However, in the absence of gfdA, osmotic stress can rescue colony growth defects and allow colonies to partially bypass the gfdA requirement in a high osmolarity glycerol pathway-dependent manner. Therefore, the findings of this study elucidate how saprophytic filamentous fungi have developed pathways distinct from those of budding yeasts to adapt to varied carbon sources and survive environmental stresses.