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.

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.

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.

Putative PmrA and PmcA are important for normal growth, morphogenesis and cell wall integrity, but not for viability in Aspergillus nidulans.

P-type Ca(2+)-transporting ATPases are Ca(2+) pumps, extruding cytosolic Ca(2+) to the extracellular environment or the intracellular Ca(2+) store lumens. In budding yeast, Pmr1 (plasma membrane ATPase related), and Pmc1 (plasma membrane calcium-ATPase) cannot be deleted simultaneously for it to survive in standard medium. Here, we deleted two putative Ca(2+) pumps, designated AnPmrA and AnPmcA, from Aspergillus nidulans, and obtained the mutants ΔanpmrA and ΔanpmcA, respectively. Then, using ΔanpmrA as the starting strain, the promoter of its anpmcA was replaced with the alcA promoter to secure the mutant ΔanpmrAalcApmcA or its anpmcA was deleted completely to produce the mutant ΔanpmrAΔpmcA. Different from the case in Saccharomyces cerevisiae, double deletion of anpmrA and anpmcA was not lethal in A. nidulans. In addition, deletion of anpmrA and/or anpmcA had produced growth defects, although overexpression of AnPmc1 in ΔanpmrAalcApmcA could not restore the growth defects that resulted from the loss of AnPmrA. Moreover, we found AnPmrA was indispensable for maintenance of normal morphogenesis, especially in low-Ca(2+)/Mn(2+) environments. Thus, our findings suggest AnPmrA and AnPmcA might play important roles in growth, morphogenesis and cell wall integrity in A. nidulans in a different way from that in yeasts.

The excreted polysaccharide of Pleurotus eryngii inhibits the foam-cell formation via down-regulation of CD36.

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 ß(1 → 3)-glucan glycosidic linkage branched at O-6 by α-D-glucose while EP digested by ß-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.

Contribution of the gas1 gene of the entomopathogenic fungus Beauveria bassiana, encoding a putative glycosylphosphatidylinositol-anchored beta-1,3-glucanosyltransferase, to conidial thermotolerance and virulence.

Beauveria bassiana is a mycoinsecticide alternative to chemicals for use in biological pest control. The fungus-insect interaction is also an emerging model system to examine unique aspects of the development, pathogenesis, and diversity of fungal lifestyles. The glycoside hydrolase 72 (GH72) family includes ß-1,3-glucanosyltransferases that are glycosylphosphatidylinositol (GPI)-anchored cell wall-modeling enzymes affecting fungal physiology. A putative B. bassiana GPI-anchored ß-1,3-glucanosyltransferase (Bbgas1) was isolated and characterized. B. bassiana targeted gene knockouts lacking Bbgas1 were affected in Congo red and salt sensitivity but displayed minor growth defects in the presence of sorbitol, SDS, or calcofluor white. Lectin and antibody mapping of surface carbohydrates revealed increased exposure of carbohydrate epitopes, including ß-1,3-glucans, in the ΔBbgas1 strain. Transmission electron micrographs revealed localized destabilization of the cell wall in ΔBbgas1 conidia, in which fraying of the outer cell wall was apparent. Heat shock temperature sensitivity profiling showed that in contrast to the wild-type parent, ΔBbgas1 conidial spores displayed decreased germination after 1 to 4 h of heat shock at temperatures >40°C, and propidium iodide exclusion assays revealed decreased membrane stability in the knockout strain at temperatures >50°C. The ΔBbgas1 knockout showed reduced virulence in Galleria mellonella insect bioassays in both topical and intrahemocoel-injection assays. B. bassiana ΔBbgas1 strains complemented with the complete Bbgas1 open reading frame were indistinguishable from the wild-type parent in all phenotypes examined. The Bbgas1 gene did not complement the phenotype of a Saccharomyces cerevisiae ß-1,3-glucanosyltransferase Δgas1 mutant, indicating that this family of enzymes likely possess discrete cellular functions.