Regulation of pseurotin A biosynthesis by GliZ and zinc in Aspergillus fumigatus.

Recently, we reported that zinc regulates gliotoxin biosynthesis via ZafA, which is a zinc-responsive transcriptional activator. From an HPLC analysis of culture media of Aspergillus fumigatus, we found a trend of decreasing gliotoxin production but increasing pseurotin A and fumagillin production in proportion to the zinc concentration. The expression of the genes involved in pseurotin A biosynthesis was upregulated under high zinc concentrations. Furthermore, upregulated expression of pseurotin A biosynthetic genes and higher production of pseurotin A were observed in the zafA deletion strain. Interestingly, the deletion of gliZ, a transcriptional activator of gliotoxin biosynthesis genes, resulted in upregulated expression of pseurotin A biosynthetic genes and increased production of pseurotin A. We detected upregulation of fumR expression in the gliZ and zafA deletion mutants. The overexpression of gliZ observed in the zafA deletion mutant resulted in the failure of the mutant to increase pseurotin A production, which is a phenotype of the zafA deletion mutant. These results suggest that ZafA sequentially regulates pseurotin A biosynthesis through GliZ. Finally, we found through a murine virulence test that the gliZ and fumR double-deletion mutants showed a delayed death rate compared with the single-deletion mutants of either gliZ or fumR. Taken together, these results suggested that the biosynthesis of gliotoxin and pseurotin A are regulated in opposite ways by zinc utilization and that each secondary metabolite is synthesized when the synthesis of another secondary metabolite fails to protect it against the defense system of the host.

AfSec1 is a signal peptidase and removes signal peptides of 1,3-ß-glucanosyltransferases in Aspergillus fumigatus.

To identify the infection mechanism of Aspergillus fumigatus, which is an opportunistic fungal pathogen, we analyzed the expression profile of the whole genome of A. fumigatus during the infection of murine macrophages. A previously reported RNA-seq data analysis showed that many genes involved in cell wall synthesis were upregulated during the infection process. Interestingly, AfSec1 (3g12840), which encodes a putative signal peptidase, was upregulated dramatically, and its putative target protein Gel1, which encodes a 1,3-ß-glucanosyltransferase, was also upregulated. Instead of the AfSec1 deletion strain, the AfSec1-ΔP strain was constructed, in which the promoter region of AfSec1 was deleted, and AfSec1 expression was not detected in the AfSec1-ΔP strain. The expression of AfSec1 was recovered by the introduction of the promoter region (the AfSec1-ΔP/P strain). The nonprocessed form of Gel1 was identified in the AfSec1-ΔP strain, which lacked the promoter, but mature forms of Gel1 were found in the wild-type and in AfSec1-ΔP/P, which was the promoter complementation strain. In the plate assay, the AfSec1-ΔP strain showed higher sensitivity against caspofungin than the wild-type. However, compared with the wild-type, the deletion strain showed no difference in the sensitivity to other antifungal drugs, such as amphotericin B and voriconazole, which inhibit different targets compared with caspofungin. The AfSec1-ΔP strain exhibited ∼20% lower levels of ß-glucan in the cell wall than the wild-type. Finally, the virulence decreased when the promoter region of AfSec1 was deleted, as observed in the murine infection test and conidia-killing assay using human macrophages and neutrophils. These results suggest that AfSec1 exerts signal peptidase activity on its target Gel1 and has an important role in fungal pathogenesis.

We identified the novel signal peptidase AfSec1 from A. fumigatus. AfSec1 which removes the signal peptide of 1,3-ß-glucanosyltransferases, is a virulence factor and is a potential target for a new antifungal drug.

Regulation of Copper Metabolism by Nitrogen Utilization in Saccharomyces cerevisiae.

To understand the relationship between carbon or nitrogen utilization and iron homeostasis, we performed an iron uptake assay with several deletion mutants with partial defects in carbon or nitrogen metabolism. Among them, some deletion mutants defective in carbon metabolism partially and the MEP2 deletion mutant showed lower iron uptake activity than the wild type. Mep2 is known as a high-affinity ammonia transporter in Saccharomyces cerevisiae. Interestingly, we found that nitrogen starvation resulted in lower iron uptake activity than that of wild-type cells without downregulation of the genes involved in the high-affinity iron uptake system FET3/FTR1. However, the gene expression of FRE1 and CTR1 was downregulated by nitrogen starvation. The protein level of Ctr1 was also decreased by nitrogen starvation, and addition of copper to the nitrogen starvation medium partially restored iron uptake activity. However, the expression of MAC1, which is a copper-responsive transcriptional activator, was not downregulated by nitrogen starvation at the transcriptional level but was highly downregulated at the translational level. Mac1 was downregulated dramatically under nitrogen starvation, and treatment with MG132, which is an inhibitor of proteasome-dependent protein degradation, partially attenuated the downregulation of Mac1. Taken together, these results suggest that nitrogen starvation downregulates the high-affinity iron uptake system by degrading Mac1 in a proteasome-dependent manner and eventually downregulates copper metabolism.

Identification of the Antidepressant Function of the Edible Mushroom Pleurotus eryngii.

Pleurotus eryngii produces various functional molecules that mediate physiological functions in humans. Recently, we observed that P. eryngii produces molecules that have antidepressant functions. An ethanol extract of the fruiting body of P. eryngii was obtained, and the extract was purified by XAD-16 resin using an open column system. The ethanol eluate was separated by HPLC, and the fraction with an antidepressant function was identified. Using LC-MS, the molecular structure of the HPLC fraction with antidepressant function was identified as that of tryptamine, a functional molecule that is a tryptophan derivative. The antidepressant effect was identified from the ethanol extract, XAD-16 column eluate, and HPLC fraction by a serotonin receptor binding assay and a cell-based binding assay. Furthermore, a forced swimming test (FST) showed that the mice treated with purified fractions of P. eryngii exhibited decreased immobility time compared with nontreated mice. From these results, we suggest that the extract of P. eryngii has an antidepressant function and that it may be employed as an antidepressant health supplement.

The Role of Zinc in Copper Homeostasis of Aspergillus fumigatus.

Copper is an essential metal ion that performs many physiological functions in living organisms. Deletion of Afmac1, which is a copper-responsive transcriptional activator in A. fumigatus, results in a growth defect on aspergillus minimal medium (AMM). Interestingly, we found that zinc starvation suppressed the growth defect of the Δafmac1 strain on AMM. In addition, the growth defect of the Δafmac1 strain was recovered by copper supplementation or introduction of the CtrC gene into the Δafmac1 strain. However, chelation of copper by addition of BCS to AMM failed to recover the growth defect of the Δafmac1 strain. Through Northern blot analysis, we found that zinc starvation upregulated CtrC and CtrA2, which encode membrane copper transporters. Interestingly, we found that the conserved ZafA binding motif 5'-CAA(G)GGT-3' was present in the upstream region of CtrC and CtrA2 and that mutation of the binding motif led to failure of ZafA binding to the upstream region of CtrC and upregulation of CtrC expression under zinc starvation. Furthermore, the binding activity of ZafA to the upstream region of CtrC was inversely proportional to the zinc concentration, and copper inhibited the binding of ZafA to the upstream region of CtrC under a low zinc concentration. Taken together, these results suggest that ZafA upregulates copper metabolism by binding to the ZafA binding motif in the CtrC promoter region under low zinc concentration, thus regulating copper homeostasis. Furthermore, we found that copper and zinc interact in cells to maintain metal homeostasis.

Response to the letter 'Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus'.

The answer to the letter 'Absent regulation of iron acquisition by the copper regulator Mac1 in A. fumigatus' has been prepared. We explained our data and showed supplementary information to answer the questions. And we respect the results of other groups first and explain the differences from our results.

The Role of Zinc in Gliotoxin Biosynthesis of Aspergillus fumigatus.

Zinc performs diverse physiological functions, and virtually all living organisms require zinc as an essential trace element. To identify the detailed function of zinc in fungal pathogenicity, we carried out cDNA microarray analysis using the model system of Aspergillus fumigatus, a fungal pathogen. From microarray analysis, we found that the genes involved in gliotoxin biosynthesis were upregulated when zinc was depleted, and the microarray data were confirmed by northern blot analysis. In particular, zinc deficiency upregulated the expression of GliZ, which encodes a Zn2-Cys6 binuclear transcription factor that regulates the expression of the genes required for gliotoxin biosynthesis. The production of gliotoxin was decreased in a manner inversely proportional to the zinc concentration, and the same result was investigated in the absence of ZafA, which is a zinc-dependent transcription activator. Interestingly, we found two conserved ZafA-binding motifs, 5'-CAAGGT-3', in the upstream region of GliZ on the genome and discovered that deletion of the ZafA-binding motifs resulted in loss of ZafA-binding activity; gliotoxin production was decreased dramatically, as demonstrated with a GliZ deletion mutant. Furthermore, mutation of the ZafA-binding motifs resulted in an increase in the conidial killing activity of human macrophage and neutrophil cells, and virulence was decreased in a murine model. Finally, transcriptomic analysis revealed that the expression of ZafA and GliZ was upregulated during phagocytosis by macrophages. Taken together, these results suggest that zinc plays an important role in the pathogenicity of A. fumigatus by regulating gliotoxin production during the phagocytosis pathway to overcome the host defense system.

The overexpression of cucumber (Cucumis sativus L.) genes that encode the branched-chain amino acid transferase modulate flowering time in Arabidopsis thaliana.

KEY MESSAGE: The overexpression of CsBCATs promotes flowering in Arabidopsis by regulating the expression of flowering time genes. The branched-chain amino acid transferases (BCATs) play an important role in the metabolism of branched-chain amino acids (BCAAs), such as isoleucine, leucine, and valine. They function in both the synthesis and the degradation of this class of amino acids. We identified and characterized the three BCAT genes in cucumber (Cucumis sativus L.). The tissue-specific expression profiling in cucumber plants revealed that CsBCAT2 and CsBCAT7 were highly expressed in the reproductive tissues, whereas CsBCAT3 expression was highly detected in the vegetative tissues. The subcellular localization patterns of three CsBCATs were observed in the mitochondria. The functional analyses of CsBCATs showed that CsBCAT2 and CsBCAT3 restored the growth of bat1Δ/bat2Δ double knockout yeast (Saccharomyces cerevisiae), and CsBCAT3 and CsBCAT7 with different substrate preferences acted in a reverse reaction. The transgenic approach demonstrated that the overexpression of the three CsBCATs resulted in early flowering phenotypes, which were associated with the upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) in a manner in which they were dependent on GIGANTEA (GI)/CONSTANS (CO) and SHORT VEGETATIVE PHASE (SVP)/FLOWERING LOCUS C (FLC) modules. Our results, which are observed in conjunction, suggest that there is an interconnection between BCAT genes that function in BCAA metabolism and the flowering time in plants.

A copper transcription factor, AfMac1, regulates both iron and copper homeostasis in the opportunistic fungal pathogen Aspergillus fumigatus.

Although iron and copper are co-ordinately regulated in living cells, the homeostatic effects of each of these metals on the other remain unknown. Here, we show the function of AfMac1, a transcriptional activator of the copper and iron regulons of Aspergillus fumigatus, on the interaction between iron and copper. In addition to the copper-specific AfMac1-binding motif 5'-TGTGCTCA-3' found in the promoter region of ctrC, the iron-specific AfMac1-binding motif 5'-AT(C/G)NN(A/T)T(A/C)-3' was identified in the iron regulon but not in the copper regulon by ChIP sequence analysis. Furthermore, mutation of the AfMac1-binding motif of sit1 eliminated AfMac1-mediated sit1 up-regulation. Interestingly, the regulation of gene expression in the iron regulon by AfMac1 was not affected by copper and vice versa AfMac1 localized to the nucleus under iron- or copper-depleted conditions, and AfMac1 was mostly detected in the cytoplasm under iron- or copper-replete conditions. Taken together, these results suggest that A. fumigatus independently regulates iron and copper homeostasis in a manner that involves AfMac1 and mutual interactions.

Correction: Necrosis-inducing peptide has the beneficial effect on killing tumor cells through neuropilin (NRP-1) targeting.

[This corrects the article DOI: 10.18632/oncotarget.8719.].

Functional characterization of the copper transcription factor AfMac1 from Aspergillus fumigatus.

Although copper functions as a cofactor in many physiological processes, copper overload leads to harmful effects in living cells. Thus, copper homeostasis is tightly regulated. However, detailed copper metabolic pathways have not yet been identified in filamentous fungi. In this report, we investigated the copper transcription factor AfMac1 ( Aspergillus fumigatusMac1 homolog) and identified its regulatory mechanism in A. fumigatus AfMac1 has domains homologous to the DNA-binding and copper-binding domains of Mac1 from Saccharomyces cerevisiae, and AfMac1 efficiently complemented Mac1 in S. cerevisiae Expression of Afmac1 resulted in CTR1 up-regulation, and mutation of the DNA-binding domain of Afmac1 failed to activate CTR1 expression in S. cerevisiae The Afmac1 deletion strain of A. fumigatus failed to grow in copper-limited media, and its growth was restored by introducing ctrC We found that AfMac1 specifically bound to the promoter region of ctrC based on EMSA. The AfMac1-binding motif 5'-TGTGCTCA-3' was identified from the promoter region of ctrC, and the addition of mutant ctrC lacking the AfMac1-binding motif failed to up-regulate ctrC in A. fumigatus Furthermore, deletion of Afmac1 significantly reduced strain virulence and activated conidial killing activity by neutrophils and macrophages. Taken together, these results suggest that AfMac1 is a copper transcription factor that regulates cellular copper homeostasis in A. fumigatus.

Chitosan dosage regimen to trap fecal oil excretion after peroral lipase inhibitor administration in mice.

This study was designed to investigate the oil entrapment and systemic oil absorption-reducing activities of chitosan. High-molecular-weight chitosan formed gel aggregates with oil and bile salts in vitro. The oil/chitosan ratio and the molecular weight of chitosan were optimized for the in vivo study, and a molecular weight >100,000 was effective in reducing the oil contamination of mouse fur. The oil/chitosan weight ratio required for effective oil entrapment was less than 13 and 5 in the in vitro and in vivo experiments, respectively. Chitosan administration was most effective during meals, and high-molecular-weight chitosan could trap and facilitate the reduction of systemic absorption of oil droplets separated by orlistat. The activity of the lipase inhibitor was not altered by chitosan as evidenced by thin layer chromatography, and orlistat was not absorbed systemically by the co-administration of chitosan.

Absorption mechanism of DHP107, an oral paclitaxel formulation that forms a hydrated lipidic sponge phase.

Paclitaxel is a most widely used anticancer drug with low oral bioavailability, thus it is currently administered via intravenous infusion. DHP107 is a lipid-based paclitaxel formulation that can be administered as an oral solution. In this study, we investigated the mechanism of paclitaxel absorption after oral administration of DHP107 in mice and rats by changing the dosing interval, and evaluated the influence of bile excretion. DHP107 was orally administered to mice at various dosing intervals (2, 4, 8, 12, 24 h) to examine how residual DHP107 affected paclitaxel absorption during subsequent administration. Studies with small-angle X-ray diffraction (SAXS) and cryo-transmission electron microscopy (cryo-TEM) showed that DHP107 formed a lipidic sponge phase after hydration. The AUC values after the second dose were smaller than those after the first dose, which was correlated to the induction of expression of P-gp and CYP in the livers and small intestines from 2 h to 7 d after the first dose. The smaller AUC value observed after the second dose was also attributed to the intestinal adhesion of residual formulation. The adhered DHP107 may have been removed by ingested food, thus resulting in a higher AUC. In ex vivo and in vivo mucoadhesion studies, the formulation adhered to the villi for up to 24 h, and the amount of DHP107 that adhered was approximately half that of monoolein. The paclitaxel absorption after administration of DHP107 was not affected by bile in the cholecystectomy mice. The dosing interval and food intake affect the oral absorption of paclitaxel from DHP107, which forms a mucoadhesive sponge phase after hydration. Bile excretion does not affect the absorption of paclitaxel from DHP107 in vivo.

Heterologous expression of a new manganese-dependent peroxidase gene from Peniophora incarnata KUC8836 and its ability to remove anthracene in Saccharomyces cerevisiae.

The white rot fungus Peniophora incarnata KUC8836 has received an attention as the greatest degrader of polycyclic aromatic hydrocarbons (PAHs), which are hazardous xenobiotics and recalcitrant pollutants. To characterize the mechanisms through which MnP degrades PAHs, heterologous expression of manganese-dependent peroxidase (MnP) gene pimp1 was performed in Saccharomyces cerevisiae BY4741 via the pGEM-T Easy vector, resulting in the recombinant plasmid pESC-URA/pimp1 containing the MnP signal peptide. MnP was significantly secreted into the culture medium with galactose as an active protein with higher efficiency (3.58 U mL-1) by transformants than by the wild-type S. cerevisiae. The recombinant MnP protein was shown to have a molecular weight of 44 kDa by western blotting analysis. With regard to enhancing the bioremediation of PAHs in the environment, anthracene was effectively degraded by the MnP encoded by pimp1, with a degradation rate of 6.5% when Tween 80 was added. In addition, the MnP activity of the transformant exhibited the highest efficiency (2.49 U mL-1) during the degradation. These results show that pimp1 might be useful for biodegradation and gene expression technologies at a transcriptional level, and genetic approaches can be improved by incorporating the highly ligninolytic gene pimp1 and the fungus P. incarnata KUC8836.

Necrosis-inducing peptide has the beneficial effect on killing tumor cells through neuropilin (NRP-1) targeting.

The therapeutic efficacy of most anti-cancer drugs depends on their apoptosis-inducing abilities. Previously, we showed that a peptide containing the mitochondrial targeting domain (MTD) found in Noxa, a BH-3 only protein of Bcl-2 family, induces necrosis. Here, a fusion peptide of neuropilin-1 (NRP-1) targeting peptide and MTD peptide, designated tumor homing motif 17:MTD (TU17:MTD), was found to induce necrosis in cancer cells in vitro and to cause the regression of tumors when intravenously injected into mice bearing subcutaneous CT26 colorectal carcinoma tumors. The necrosis within tumor tissues was evident upon administering TU17:MTD. TU17:MTD penetrated into tumor cells by targeting to Neuropilin-1, which could be blocked by anti-NRP-1 antibody. The efficacy of TU17:MTD on tumor regression was higher than that of TU17:D(KLAKLAK)2, a fusion peptide of NRP-1 targeting peptide and a pro-apoptotic peptide. The necrotic cell death within tumor tissues was evident at day 1 after administering TU17:MTD systemically. Transplanted subcutaneous substantially reduced in size within two weeks and 5 days, respectively, with no apparent side effects. Together, these results propose that the pro-necrotic peptide MTD may present an alternative approach for development of targeted anti-cancer agents.

Identification of ferrichrome- and ferrioxamine B-mediated iron uptake by Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen for immunocompromised patients, and genes involved in siderophore metabolism have been identified as virulence factors. Recently, we identified the membrane transporters sit1 and sit2, which are putative virulence factors of A. fumigatus; sit1 and sit2 are homologous to yeast Sit1, and sit1 and sit2 gene expression was up-regulated after iron depletion. When expressed heterologously in Saccharomyces cerevisiae, sit1 and sit2 were localized to the plasma membrane; sit1 efficiently complemented ferrichrome (FC) and ferrioxamine B (FOB) uptake in yeast cells, whereas sit2 complemented only FC uptake. Deletion of sit1 resulted in a decrease in FOB and FC uptake, and deletion of sit2 resulted in a decrease in FC uptake in A. fumigatus It is of interest that a sit1 and sit2 double-deletion mutant resulted in a synergistic decrease in FC uptake activity. Both sit1 and sit2 were localized to the plasma membrane in A. fumigatus The expression levels of the sit1 and sit2 genes were dependent on hapX under low-but not high-iron conditions. Furthermore, mirB, and sidA gene expression was up-regulated and sreA expression down-regulated when sit1 and sit2 were deleted. Although sit1 and sit2 failed to affect mouse survival rate, these genes affected conidial killing activity. Taken together, our results suggest that sit1 and sit2 are siderophore transporters and putative virulence factors localized to the plasma membrane.

The vest-collar as a rodent collar to prevent licking and scratching during experiments.

Various types of restraint collars have been used for research animals, and the Elizabethan collar (E-collar) is the most commonly used. However, animals can be choked by the E-collar or they tend to remove it; furthermore, repeated rubbing and scratching of the collar may chafe the neck. We developed a new restraint collar with a vest to overcome these limitations. The vest-collar (V-collar) can be worn similarly to a vest, in contrast to the E-collar, which is fixed around the neck. A cone-shaped collar is attached to the vest in the V-collar and is made of Eva foam to surround the chest softly, accompanied by a transparent polyvinyl chloride (PVC) film for visibility. To evaluate the performance of the V-collar, we conducted experiments with mice wearing the V-collar and the E-collar. Both groups showed normal weight gain and food intake. Glucose and stress hormone levels showed no significant differences, and no stress-associated leukocyte profiles were observed during the experiments. However, despite the short experimental duration, more than half of the mice in the E-collar group showed injury to the skin on the neck, with increased thickness of the epidermal and keratin layers. Moreover, inflammatory cell counts were higher in the E-collar group than in the V-collar group. In conclusion, the V-collar, in contrast to the E-collar, does not cause skin injuries in animals and is thus beneficial for animals and investigators. Investigators can effectively use the V-collar to enhance laboratory animal welfare.

Rck1 promotes pseudohyphal growth via the activation of Ubp3 phosphorylation in Saccharomyces cerevisiae.

Previously, we reported that Rck1 up-regulates Ras2 and pseudohyphal growth of Saccharomyces cerevisiae. Here, we further investigate the involvement of Rck1 in the activation of pseudohyphal growth. Rck1 activated phosphorylation of the deubiquitinase Ubp3 through a direct protein interaction between Rck1 and Ubp3. The N-terminal Bre5 binding region of Ubp3 physically interacted with Rck1, and Ubp3 and Rck1 co-precipitated. Overexpression of UBP3 using a high-copy plasmid resulted in the upregulation of Ras2, and deletion of UBP3 blocked the upregulation of Ras2 by RCK1 overexpression. Treatment with the proteasome inhibitor MG132 resulted in accumulation of Ras2, indicating that Rck1 is involved in Ras2 degradation in a proteasome-dependent manner. Furthermore, deletion of UBP3 blocked the upregulation of FLO11, a flocculin required for pseudohyphal and invasive growth induced by RCK1 overexpression in S. cerevisiae. Taken together, these results demonstrate that Rck1 promotes S. cerevisiae pseudohyphal growth via the activation of Ubp3 phosphorylation.

Physical interaction between Sit1 and Aft1 upregulates FOB uptake activity by inhibiting protein degradation of Sit1 in Saccharomyces cerevisiae.

Previously, we reported that Aft1 regulates Sit1 by modulating the ubiquitination of Sit1 in Saccharomyces cerevisiae. Here, we report the function of the physical interaction between Sit1 and Aft1 in ferrioxamine B (FOB) uptake. The interaction between Sit1 and Aft1 induced protein localization of Sit1 to the plasma membrane, and more Sit1 was detected in the plasma membrane when Sit1 and Aft1 were coexpressed compared with Sit1 expression alone. The MSN5-deletion mutant, which failed to translocate Aft1 to the cytosolic compartment, showed lower FOB uptake activity than the wild type. However, higher free iron uptake activity was detected in the MSN5-deletion mutant. Furthermore, the strain transformed with AFT1-1(up) plasmid, which failed to regulate Aft1 via iron concentration and accumulated Aft1 in the nucleus, showed lower FOB uptake activity. The Aft1 Y179F mutant, which contained a tyrosine residue that was changed to phenylalanine, failed to interact physically with Sit1 and showed more degradation of the Sit1 and, ultimately, lower FOB uptake activity. Additionally, we found that MG132 and PMSF, which are inhibitors of proteasomes and serine proteases, respectively, increased the Sit1 protein level. Taken together, these results suggest that the protein-protein interaction between Sit1 and Aft1 is an important factor in the FOB uptake activity of Sit1.

Identification of high-affinity copper transporters in Aspergillus fumigatus.

We investigated the copper metabolism of Aspergillus fumigatus, which has not been characterized well. We cloned the putative copper transporters ctrA2 and ctrC from A. fumigatus and investigated the functions of these transporters in copper metabolism. Four putative copper transporters were identified in the A. fumigatus genome; ctrA2 and ctrC complemented CTR1 functionally and localized to the plasma membrane in Saccharomyces cerevisiae. ctrA2 and ctrC single-deletion mutants and a double-deletion mutant of ctrA2 and ctrC were constructed in A. fumigatus. The ctrA2 and ctrC double-deletion mutant exhibited a growth defect on Aspergillus minimal medium (AMM) supplemented with bathocuproine disulfonic acid (BCS) and was sensitive to H2O2. Furthermore, the deletion of ctrA2 and ctrC reduced superoxide dismutase (SOD) activity, laccase activity, and intracellular copper contents. The activities of the ctrA2 and ctrC genes were up-regulated by BCS treatment. In addition, the deletion of ctrA2 up-regulated ctrC and vice versa. ctrA2 and ctrC were localized to the A. fumigatus plasma membrane. Although ctrA2 and ctrC failed to affect the mouse survival rate, these genes affected conidial killing activity. Taken together, these results indicate that ctrA2 and ctrC may function as membrane transporters and that the involvement of these genes in pathogenicity merits further investigation.