Functional characterization of genes encoding cadmium pumping P_1B-type ATPases in Aspergillus fumigatus and Aspergillus nidulans

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 P_1B-type ATPases. These ATPases usually pump Ag⁺, Cu⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Ni²⁺, Pb²⁺, or Zn²⁺ from the cell (8, 9). Some P_1B-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 P_1B-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 P_1B-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 P_1B-type ATPases to metal homeostasis can promote research on these pumps as antifungal target.

The function of the fungal P_1B-type ATPases has been extensively studied in S. cerevisiae and Candida albicans. S. cerevisiae has a copper (Ccc2) and cadmium (Pca1) P_1B-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 P_1B-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, P_1B-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 P_1B-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 P_1B-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 P_1B-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.

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. P_1B-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.

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.