Several P1B-type ATPases are important Cd2+/Cu2+ pumps in Aspergillus species, and they are tightly associated with the heavy metal stress tolerance of these ascomycetous fungi. To better understand the roles of the two P1B-type ATPases, Aspergillus nidulans CrpA Cd2+/Cu2+ pump (orthologue of the Candida albicans Crp1 Cd2+/Cu2+ pump) and Aspergillus fumigatus PcaA Cd2+ pump (orthologue of the Saccharomyces cerevisiae Pca1 Cd2+ pump), we have generated individual mutants and characterized their heavy metal susceptibilities. The deletion of CrpA in A. nidulans has led to the increased sensitivity of the fungus to stresses induced by Zn2+, Fe2+, or the combination of oxidative-stress-inducing menadione sodium bisulfite and Fe3+. Heterologous expression of A. fumigatus PcaA in the S. cerevisiae pca1 deletion mutant has resulted in enhanced tolerance of the yeast to stresses elicited by Cd2+or Zn2+ but not by Fe2+/Fe3+ or Cu2+. Mammalian host immune defense can attack microbes by secreting Zn2+ or Cu2+, and the oxidative stress induced by host immune systems can also disturb metal (Cu2+, Fe2+, and Zn2+) homeostasis in microbes. In summary, PcaA and CrpA can protect fungal cells from these complex stresses that contribute to the virulence of the pathogenic Aspergillus species. Moreover, due to their presence on the fungal cell surface, these P1B-type ATPases may serve as a novel drug target in the future.
Mammalian host immune defense disrupts heavy metal homeostasis of fungal pathogens. P1B-type ATPase of Aspergillus fumigatus and Aspergillus nidulans may help to cope with this stress and serve as virulence traits. In our experiments, both A. nidulans Cd2+/Cu2+ pump CrpA and A. fumigatus Cd2+ pump PcaA protected fungal cells from toxic Zn2+, and CrpA also decreased Fe2+ susceptibility most likely indirectly. In addition, CrpA protected cells against the combined stress induced by the oxidative stressor menadione and Fe3+. Since P1B-type ATPases are present on the fungal cell surface, these proteins may serve as a novel drug target in the future.
Aspergillus fumigatus is one of the most prevalent filamentous fungal pathogens, causing life-threatening invasive aspergillosis in immunocompromised patients (1). Contribution of other Aspergillus species than A. fumigatus to fungal infections is also substantial: Aspergillus nidulans, known as a model organism in mycology, is responsible for the majority of invasive aspergillosis accompanied by chronic granulomatous disease (2-4). Due to the limitations of current therapies new strategies to increase therapeutic efficacy against aspergilli must be developed. P-type ATPases are considered as anti-fungal targets since they are easily accessible on the cell surface, and many play a pivotal role in the virulence of microorganisms (5-7).
P-type ATPases are a large and unique family of membrane proteins involved in various transport processes in nearly all cells. These ion pumps are quite widespread in eukaryotes, including fungi, and contribute to important physiological processes including the maintenance of ion homeostasis, membrane potential, and lipid-bilayer asymmetry, as well as the detoxification of transient metals (8). This latter phenomenon is attributed to P1B-type ATPases. These ATPases usually pump Ag+, Cu+, Cd2+, Co2+, Cu2+, Fe2+, Ni2+, Pb2+, or Zn2+ from the cell (8, 9). Some P1B-type ATPases are important virulence traits in bacteria and even in fungi (10-16). Their contribution to virulence is sometimes unclear. In the case of bacterial Fe2+-ATPases, it is assumed that Fe2+ efflux protects cells against iron overload, e.g., when bacteria escape from phagosomes to the relatively iron-rich cytosol (11). Alternatively, Fe2+-ATPases protect cells against toxic Fe2+ liberated within the cells under oxidative stress (11). Macrophages secrete Cu2+ and superoxide anion into the phagosomes to kill the embedded microbes, which may explain why microbial Cu2+ pumps like A. fumigatus CrpA or Aspergillus flavus CrpA and CrpB act as a virulence factor (13, 14).
Here, we have characterized the function of two P1B-type Cd2+ ATPases: CrpA (17, 18) of A. nidulans as an emerging opportunistic fungal pathogen (3) and PcaA (15, 19) of the well-known opportunistic human pathogen A. fumigatus. We show that the function of these Cd2+ pumps goes beyond protecting cells from this toxic heavy metal. They transport ions other than Cd2+ (e.g., Zn2+) and, due to the tight coupling between the metabolism of different metal ions, they may even affect the homeostasis of ions (e.g., Fe2+/Fe3+) that they are unlikely to transport. These properties of P1B-type microbial ATPases may explain why they have been identified as a virulence trait in many microorganisms (10-16). In the case of CrpA, we studied gene deletion strains to characterize the function of this protein. Recently, Bakti et al. (15) demonstrated that deletion of pcaA reduced the virulence of A. fumigatus in the Galleria mellonella infection model; however, the gene deletion mutant showed only increased Cd2+ but not Cu2+, Fe2+, or Zn2+ sensitivity (15). We speculated that, when A. fumigatus mutants were tested, the consequences of deletion/overexpression of the pcaA gene might have been masked or counteracted by elements of the heavy metal detoxification system other than PcaA. Therefore, we expressed A. fumigatus pcaA in Saccharomyces cerevisiae to study its functions in a host cell different from A. fumigatus. Understanding the contribution of P1B-type ATPases to metal homeostasis can promote research on these pumps as antifungal target.
RESULTS AND DISCUSSION
The function of the fungal P1B-type ATPases has been extensively studied in S. cerevisiae and Candida albicans. S. cerevisiae has a copper (Ccc2) and cadmium (Pca1) P1B-type ATPase. Ccc2 belongs to the 1B-1 subfamily (9); it localizes in the trans-Golgi membrane and provides Cu2+ to the multicopper ferroxidase Fet3, thus indirectly participating in iron uptake (20). Pca1 is a member of the 1B-2 subfamily (9). In addition to the Cd2+ detoxification, Pca1 also contributes to the Cu2+ tolerance by sequestering Cu2+ in its Cys-rich N-terminal region and may also play a role in iron homeostasis (21, 22). C. albicans has two copper P1B-type ATPases, Crp1 (Crd1) and Ccc2 (23-25). Crp1 functions as a Cu2+, Cd2+, and Ag+ pump (23, 24). C. albicans Ccc2, like its S. cerevisiae orthologue, localizes in the Golgi membrane and indirectly affects iron uptake (25).
The Aspergillus genomes studied (275 strains of 256 species) encode two to four, even up to eight, P1B-type ATPase genes (Table S1; Fig. 1). Each strain has at least one (maximum five) Crp1 (CrpA) and one (maximum two) Ccc2 (CtpA) orthologues (Table S1; Fig. 1). Interestingly, the Pca1 orthologue PcaA is present in only 109 strains (Table S1; Fig. 1) (26). A. nidulans has two P1B-type ATPases, CrpA (orthologue of C. albicans Crp1) and YgA (orthologue of C. albicans Ccc2) (Table S1; Fig. 1). CrpA is responsible for Cu2+ and Cd2+ tolerance and can pump Ag+ (17, 18). YgA is involved in copper compartmentalization and provides Cu2+ for conidial pigmentation for the activity of the developmental phenol oxidase, IvoB (27). The genome of A. fumigatus (Af293) encodes three P1B-type ATPases (Table S1; Fig. 1). The C. albicans Crp1 orthologue CrpA functions as a Cu2+ and Zn2+ pump (13, 28). The Afu4g12620 gene encodes a putative copper-transporting ATPase (29), which is the orthologue of C. albicans Ccc2 (Fig. 1; Table S1). PcaA (orthologue of S. cerevisiae Pca1) is involved in Cd2+ detoxification (15). PcaA was not revealed to be essential for wild-type-like Cu2+, Fe2+, or Zn2+ tolerance, but deletion of pcaA decreased, while overexpression of it increased, oxidative stress tolerance elicited by menadione sodium bisulfite (MSB) (15). The A. fumigatus Af293 strain, where pcaA is highly active, showed significantly stronger virulence in the mouse infection system than other wild-type strains with small or negligible pcaA activity (19). The deletion of pcaA also attenuated the virulence of A. fumigatus in the G. mellonella infection model. This phenotype was explained by the altered oxidative stress tolerance of the gene-deletion mutant, and this lack of PcaA may influence the activity of other metal homeostasis proteins involved in virulence (15). Note that the genome of A. fumigatus A1163 contains only C. albicans Crp1 and Ccc2 orthologues but no S. cerevisiae Pca1 orthologue (Table S1). In fact, Barber et al. (30) found that unlike crp1 and ccc2, which are part of the core genome of A. fumigatus, pcaA is an accessory gene occurring in only 2.67% of the studied 300 isolates. The data available at FungiDB (https://fungidb.org) also support this observation: Out of 879 whole genome sequenced isolates where the sample type (clinical vs environment) was clearly identifiable, only 32 (3.64%) isolates possessed the pcaA gene (Table S2). Importantly, 30 of the isolates having pcaA were clinical isolates, which represents a significant enrichment of the pcaA harboring isolates within clinical isolates (Fisher exact test; P = 0.0001767; Table S2). This enrichment suggests that although PcaA is not essential for virulence, possessing this gene can be beneficial for clinical isolates.
In order to better understand how PcaA contributes to the oxidative stress tolerance and virulence of A. fumigatus Af293, we expressed pcaA in S. cerevisiae and examined the phenotype of the mutant. It was hoped that this approach would reveal properties of PcaA that had not previously been found using pcaA gene deletion/overexpression strains. Moreover, to gain a broader understanding of the function of Cd2+-transporting P1B-type ATPases, we also investigated the function of A. nidulans CrpA, another known cadmium pump of aspergilli.
Regarding CrpA, we found that all the tested A. nidulans ΔcrpA strains were more sensitive to ZnSO4 treatment than the reference strain (Fig. 2; Table 1), suggesting the involvement of this pump in Zn2+ efflux, as was also found with A. fumigatus CrpA (28). The gene deletion strains showed increased Fe2+ sensitivity as well (Fig. 3; Table 1). In contrast, Fe3+ tolerance from the studied four strains and their MSB-elicited oxidative stress tolerance did not differ substantially from one another (Table 2; Fig. S1A and S1B). Interestingly, MSB and Fe3+ stresses showed an antagonistic effect when they were combined (Table 2; Fig. S1C): FeCl3 at 3 mM concentration completely inhibited the growth of the fungus. However, cultures were able to grow when MSB and 3 mM FeCl3 were added together (Table 2; Fig. S1C). MSB increases superoxide production in cells; superoxide reduces Fe3+ to Fe2+ and also destroys Fe-S cluster proteins (31). The buffered superoxide production due to the high Fe3+ concentration as well as the reduced Fe3+ levels due to Fe3+ - Fe2+ reduction and increased iron utilization may explain the observed antagonistic effect. When FeCl3 was applied at 3.25 mM concentration, a clear difference was found between the mutants and the reference strain: All the gene deletion strains showed decreased tolerance to the combined MSB-FeCl3 stress compared to the reference strain (Table 2; Fig. 4). These data suggest that CrpA contributes to iron metabolism as well. CrpA may pump out the excess Fe2+ from the cells or more likely mediate iron metabolism indirectly. Oxidative stress (induced by MSB) can disturb metal ion homeostasis and may liberate potentially harmful Zn2+ (32) and Cu2+. CrpA, by pumping these ions out of the cells, protects the fungus from this stress. Emerging data show that there is a tight association between the metabolism of different transition metals in fungi, in addition to the copper dependence of iron uptake via the reductive iron assimilation pathway, which has been observed in several fungi (20, 25, 33). It has also been shown that an increase in intracellular copper levels can elevate the iron content in A. fumigatus cells (34). Iron availability also regulates zinc metabolism mediated by the transcription factor ZafA (35), and in line with this, iron starvation has been shown to upregulate the vacuolar zinc transporter ZrcA (important in removing excess zinc from cytosol) and downregulate the zinc importer ZrpB in A. fumigatus (36). In addition, ZafA upregulates CtrC and CtrA2 copper transporters at low zinc concentrations (37). Increased extracellular Fe2+ or Fe3+ levels may disrupt the fine-tuned coordination of zinc-iron and/or copper-iron metabolism, resulting in the need for CrpA-mediated Zn2+ and/or Cu2+ efflux.
TABLE 1 Effect of ZnSO4 or FeSO4 on the growth of ΔcrpA (MKL 5, MKL 10, and MKL 14) and reference (TNJ 36) A. nidulans strainsd
Colony diameters of 5- (ZnSO4 treatment) or 3- (FeSO4 treatment) day-old cultures (mean ± SD; n = 3) are presented. Data were statistically analyzed with a two-way ANOVA followed by Tukey post-hoc test (P < 0.05).
Significant difference between the mutant and the reference strain.
Significant difference between the untreated and the appropriate stress-treated cultures.
Significant interaction between the effect of treatment (treated vs untreated) and gene deletion (mutant vs reference strain) in one (ZnSO4 or FeSO4 treatment) experiment.
TABLE 2 Effect of FeCl3, MSB, and combined FeCl3 and MSB treatments on the growth of ΔcrpA (MKL 5, MKL 10, and MKL 14) and reference (TNJ 36) A. nidulans strainsd
Colony diameters of 5-day-old cultures (mean ± SD; n = 3) are presented. Data were statistically analyzed with a two-way ANOVA followed by Tukey post-hoc test (P < 0.05).
Significant difference between the mutant and the reference strain.
Significant difference between the untreated and stress-treated cultures.
Significant interaction between the effect of treatment (treated vs untreated) and gene deletion (mutant vs reference strain).
Expression of the Afu-pcaA gene in S. cerevisiae increased its CdCl2 tolerance relative to the Sc-pca1 null mutant, as was expected (Fig. 5 and 6). No differences between the S. cerevisiae pca1+ and pca1- strains were found, in line with the fact that Pca1 does not function as Cd2+ transporter in laboratory strains due to a missense mutation in its gene (22). The Afu-pcaA gene also increased ZnSO4 tolerance of the yeast (Fig. 5 and 6). The involvement of PcaA in zinc homeostasis was not recorded when a gene deletion strain was studied (15). It is possible that under the studied culturing conditions, some functions of PcaA were replaced by other zinc-exporting proteins like ZrcA (36) and CrpA (28). The involvement of PcaA in zinc homeostasis may explain how this cadmium pump contributes to the oxidative stress tolerance and virulence of A. fumigatus (15, 19): Mammalian hosts use Zn2+ to protect the mucosal surface against microbes (38), and oxidative stress can disrupt metal ion homeostasis, leading to the release of toxic Zn2+ within cells (39).
Regarding FeCl3, FeSO4, or MSB tolerance, no changes were recorded (Fig. 4). Interestingly, although S. cerevisiae Pca1 contributes to the Cu2+ detoxification by sequestering Cu2+ at its Cys-rich N-terminus region (21) and the Cys-rich N-terminus also occurs in A. fumigatus PcaA, expression of the pcaA gene in S. cerevisiae (Fig. 5) or the deletion of the pcaA gene in A. fumigatus (15) did not alter the Cu2+ susceptibility of the fungus. MSB and FeCl3 and MSB and CuCl2 have antagonistic effects on S. cerevisiae survival (Fig. 5). The observation that both iron and copper could suppress the growth inhibitory effect of MSB supports the hypothesis that these transition metals may act as redox buffers during MSB-induced oxidative stress.
Zinc and iron have been considered key players in host-pathogen interactions. Hosts sequester these essential metal ions from microbes as part of nutritional immunity (40). Not surprisingly, efficient iron and zinc ion acquisition mechanisms are essential for the in vivo virulence of pathogens (40, 41). In addition, microbes must cope with metal toxicity during infection. Mammalian hosts release Zn2+ onto the mucosal surface, which is toxic to pathogens, especially in combination with Mn2+ (38) or iron (36) withdrawal. Macrophages secrete Cu2+ into the phagosomes to kill the ingested microbes (13, 14). Moreover, iron-limited conditions of phagosomes and superoxide anion secreted into the phagosomes enhance copper toxicity further (34). Microbes may also suffer from iron/zinc overload when they escape from phagosomes to the cytosol (11). Besides these effects, microbes frequently must cope with oxidative stress during infection. Oxidative stress disturbs metal (Cu2+, Fe2+, Mn2+, and Zn2+) homeostasis, leading to metal toxicity. P1B-type ATPases, by secreting different metal ions, can protect microbes from these stresses, which explains why these ATPases are important virulence traits (10-16-19).
The A. nidulans CrpA and A. fumigatus PcaA cadmium pumps can protect cells from zinc toxicity (Fig. 2, 5 and 6; Table 1), and CrpA also contributes to the Cu2+ detoxification (17, 18). Both ATPases increase the oxidative stress tolerance of the fungus (Table 2; Fig. 3) (15) presumably by stabilizing the perturbed metal ion homeostasis. In addition, CrpA can also reduce iron toxicity (Fig. 3 and 4; Tables 1 and 2) probably indirectly through the secretion of Cu2+ and/or Zn2+. Thus, these cadmium pumps may protect cells from dangerous metal ions they can pump (which are not limited to Cd2+) and may even reduce the toxicity of those they cannot pump due to the tight coupling of the metabolism of different metal ions (33-36). These properties of the two ATPases can contribute to the virulence of both A. nidulans (3) and A. fumigatus (1) and therefore represent potential targets for antifungal therapy.
MATERIALS AND METHODS
Strains and culture conditions
Strains listed in Table 3 were used in this study. Aspergillus strains were maintained on Barratt’s minimal nitrate agar plates, supplemented with pyridoxine, at 37°C (42). Conidia, freshly isolated from 6-day-old cultures, were used in all experiments.
S. cerevisiae strains were selected and maintained on SC-dropout agar plates without uracil (SC plates) (45). Cultures were incubated at 30°C for 2 days, and these 2-day-old cultures were used to study their stress tolerance.
Construction of S. cerevisiae pca1Δ and pca1Δ:Afu-pcaA strains
For amplification of A. fumigatus pcaA, total RNA was isolated from A. fumigatus Af293 cultures as described by Kurucz et al. (19) and reverse transcribed with First Strand cDNA Synthesis Kit (Thermo Scientific; Waltham, MA, USA) following the manufacturer’s protocol. The pcaA cDNA was amplified with the primer pair listed in Table 4.
Checking primers S. cerevisiae pca1Δ and pca1Δ:Afu-pcaA strains:
Genetic modification of S. cerevisiae BY4742 was carried out using the MoClo Yeast Tool Kit CRISPR/Cas9 system (Addgene, Watertown, MA, USA). The CRISPR guides and repair DNA oligos were designed using the Benchling online software (http://www.benchling.com). The single plasmid that expresses both the Cas9 nuclease and guide RNA cassette required for targeting the desired pca1 locus was developed according to Lee et al. (46). The DNA oligos used are listed in Table 4. Both repair DNA fragments contained the same 60-bp-long flanking regions homologous to the upstream and downstream parts of S. cerevisiae pca1 ORF. The pca1Δ repair DNA fragment contained in-frame stop codons as insert. The insert of the pca1Δ:Afu-pcaA repair DNA fragment consisted of A. fumigatus pcaA cDNA ORF, keeping the original S. cerevisiae pca1 promoter. Repair DNA fragments were created with PCR using Dream Taq polymerase (Thermo Scientific, Waltham, MA, USA). S. cerevisiae BY4742 was transformed using the lithium acetate method (46). The pca1 knock-out and Afu-pcaA-expressing mutants were verified by PCR using primer pairs listed in Table 4.
Stress susceptibility tests
In the case of A. nidulans strains, Barratt’s minimal nitrate plates containing pyridoxine and supplemented with 0–35 mM ZnSO4, 0–3.25 mM FeCl3, or 0–0.3 mM menadione sodium bisulfite were point-inoculated with 5 µL fresh conidia suspension (105 conidia/mL) and were incubated at 37°C for 5 days. The diameter of the colonies was recorded and used to characterize stress susceptibility. In some cases, MSB (0–0.25 mM final concentration) was added to media containing either 3 mM or 3.25 mM FeCl3. To test FeSO4 susceptibility, conidia were harvested in Barratt’s minimal nitrate medium, and the suspensions (105 conidia/mL) were incubated for 8 h at 37°C. Pre-incubated (germinated) conidia were point-inoculated onto freshly prepared agar plates containing 0, 8, or 10 mM FeSO4, and the survival of the strains was monitored after 3 days of incubation at 37°C. Importantly, Fe2+ can be quickly (within hours) oxidized to Fe3+ under aerobic conditions. This experimental setup allowed us to test the effect of Fe2+ on the freshly formed hyphae. All experiments were carried out with three biological replicates. Since genetically manipulated mutants may harbor unexpected mutations and/or the genetic manipulation may have unexpected consequences that may affect their growth and stress sensitivity, three independent crpA gene deletion strains were studied, and only their shared phenotypes were discussed.
In the case of S. cerevisiae strains, overnight cultures were grown in 5 mL aliquots of SC-dropout broth without uracil (SC broth) at 30°C and 220 rpm. Aliquots (10 mL) of SC broth inoculated with overnight cultures (starting OD600 = 0.1) were incubated at 30°C and 220 rpm until the OD600 reached 0.4 value (approximately 4 h). Then, cultures were diluted (1×, 10×, 100×, 1,000×, or 10,000×) with sterile water, and 5 µL from each dilution was point inoculated on SC plates, and the SC plates were supplemented with 0.1 mM CdCl2, 0.4 mM MSB, 1 mM and 3 mM CuCl2, 6 mM FeCl3, 32 mM ZnSO4, or 0.4 mM MSB plus 6 mM FeCl3. Cultures were incubated at 30°C for 5 days. In the case of Fe2+ tolerance tests, cultures, after reaching the OD600 = 0.4 value, were either supplemented or not with FeSO4 (1.2 M final concentration) and were incubated for 0.5 h at 30°C and 220 rpm before point inoculation on SC plates. All experiments were carried out with three biological replicates.
The CdCl2 and ZnSO4 susceptibilities of S. cerevisiae strains were also tested using a broth microdilution method in SC broth in line with the CLSI standard M27-A3 guideline (47). Tests were performed in 96-well microtiter plates at 30°C. Each well contained 200 µL medium and was inoculated with approximately 103 cells. The final metal concentrations were 0.8, 1.2, and 1.6 mM in the case of ZnSO4 and 0.2, 0.3, and 0.4 µM in the case of CdCl2. Note that due to the small inoculum size, the tested metal concentrations had to reduce markedly relative to those applied in agar plate tests. All strains were tested on four independent plates.
In silico analyses of P1B-type ATPase orthologues in Aspergillus
Putative P1B-type ATPase orthologues were collected from the JGI MycoCosme database (https://mycocosm.jgi.doe.gov/mycocosm/home) using the blastp algorithm with default settings and S. cerevisiae Ccc2 as query sequence. Only hits with more than 800-bit score value were involved in the analysis. In the cases of A. nidulans and A. fumigatus (both Af293 and A1163), there were no hits in the bit score range of 400–800. The evolutionary history of the collected Aspergillus proteins as well as S. cerevisiae Ccc2, Pca1, and C. albicans Ccc2, Crp1 proteins was inferred by the maximum likelihood method and JTT matrix-based model (48) using the MEGA11 software (49).
Research was financed by the European Union and the European Social Fund through project EFOP-3.6.1-16-2016-00022, by the National Research, Development, and Innovation Office (Hungary) projects NN125671 and K131767, and by the New National Excellence Program (ÚNKP-21-3) of the Ministry for Innovation and Technology in Hungary. Project no. TKP2021-EGA-20 (Biotechnology) has been implemented with the support provided from the National Research, Development, and Innovation Fund of Hungary, financed under the TKP2021-EGA funding scheme.
The work at UW-Madison was supported by the Food Research Institute. The work was also supported by the Korea Innovation Foundation grant funded by the Ministry of Science and ICT (2022-DD-RD-0574-02) to M.-K. Lee.
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