1. Introduction
Copper (Cu) is a transition metal essential for all living systems from bacteria to eukaryotes. Cu ions serve as important catalytic cofactors in redox chemistry for proteins that are required for growth and development. Cu-requiring proteins are involved in a variety of biological processes such as respiration, protection against oxidative stress, pigment formation, neuro-transmitter biosynthesis, peptide amidation, iron transport, and connective tissue maturation [
1]. Cu has an ability to redox cycle between two forms: the extracellular oxidized form (Cu
2+) and the intracellular reduced form (Cu
+) and that is the reason for its usefulness as a cofactor. However, the Cu
+ ↔ Cu
2+ reversible transition can also transform copper into a toxic compound through a Fenton-like reaction which generates hydroxyl radicals by reacting with hydrogen peroxide, a natural by-product of aerobic respiration [
2]. These hydroxyl radicals damage DNA by inducing strand breaks, oxidizing nucleoside bases and inactivating iron–sulfur cluster-containing enzymes, a process shared with hydrogen peroxide [
3]. Cu can also cause mismetallation, where it replaces metal cofactors in proteins, rendering them inactive [
4]. Because of this dual role, copper being at the same time essential and toxic, all living organisms have developed mechanisms to accurately tune its homeostasis [
5]. Cu homeostasis in fungi is mediated by the transcriptional regulation of genes participating in Cu acquisition, mobilization, and sequestration [
1]. In response to high toxic extracellular Cu concentrations, fungal transcription factors regulate the expression of genes responsible for the sequestration and efflux of excess Cu [
6]. In humans, infection leads to increased concentrations of Cu in the serum, and macrophages accumulate high phagolysosomal Cu levels to destroy ingested pathogens [
7,
8,
9,
10].
The Cu-buffering system in the human pathogenic mold
Aspergillus fumigatus relies primarily on AceA-dependent transcriptional activation of CrpA, encoding a Cu
+ P-type ATPase highly conserved in eukaryotes and prokaryotes [
5,
9,
11,
12,
13]. The AceA–CrpA axis is conserved in additional fungi, including
Aspergillus nidulans [
14],
Fusarium oxysporum [
15], and
Candida albicans [
16]. CrpA actively transports excess Cu from the cytoplasm to the extracellular environment. CrpA contains eight transmembrane domains, a conserved CPC (Cys–Pro–Cys) Cu translocation motif in the sixth transmembrane segment and cysteine-rich metal-binding motifs in the cytoplasmic N-terminal, and is apparently localized at the cell surface [
11]. Deletion mutants of
aceA or
crpA in
A. fumigatus are hypersensitive to Cu in-vitro. They accumulate higher intracellular Cu levels and are more susceptible to killing by macrophages. In a mouse infection model of invasive pulmonary aspergillosis, these mutants display reduced growth and virulence [
9,
11]. CrpA overexpression in the
aceA null background re-establishes a wild-type phenotype, confirming that CrpA is the major effector target gene of AceA [
9].
This study aimed at finding fungal-unique domains in the A. fumigatus Cu exporter CrpA, and analyze their function. We used a bioinformatics approach to identify two CrpA fungal-specific regions that we studied by deletion/replacement, subcellular localization, Cu sensitivity in vitro, killing by mouse alveolar macrophages, and virulence in a mouse infection model. Deleting CrpA amino acids 1–211 moderately increased Cu sensitivity without altering the localization of the protein. Deleting CrpA amino acids 542–556 strongly increased Cu sensitivity and retention of the protein to the ER. Surprisingly, deleting CrpA amino acids 1–211 or replacement of amino acids 542–556 did not affect A. fumigatus virulence in a mouse model of infection, suggesting a more complex role for the involvement of this Cu transporter in the progression of invasive pulmonary aspergillosis.
2. Materials and Methods
Strains and media.
A. fumigatus CEA17 KU80 [
17], was used to generate the strains described in this study [
17]. This commonly used laboratory strain is derived from a patient isolate and has been engineered to be deficient for non-homologous end joining. It therefore has high rates of homologous recombination, making it ideal for genetic manipulation. For routine culture, strains were grown on yeast-extract-rich solid medium (YAG) containing 0.5% (
w/
v) yeast extract, 1% (
w/
v) glucose, 10 mM MgSO
4, supplemented with 0.1% (
v/
v) trace elements solution, and 0.2% (
v/
v) vitamin mix. After incubation for 48 h at 37 °C, conidia were harvested in 0.02% (
v/
v) Tween-20, resuspended in double-distilled water (DDW), and counted with a hemocytometer. Conidial stocks were stored at 4 °C for no longer than two weeks. For experiments, strains were grown on defined minimal medium and vitamins (MMV), containing 70 mM NaNO
3, 1% (
w/
v) glucose, 12 mM potassium phosphate pH 6.8, 4 mM MgSO
4, supplemented with vitamins, trace elements, and 0.1% (
w/
v) uracil/uridine (UU) as needed. The strains used in this study and their construction are described in
Supplementary Table S1 and the Supplementary Materials section.
Structural and bioinformatic analysis of A. fumigatus CrpA. A model structure of
A. fumigatus CrpA was built using AlphaFold [
18] with templates searched against the pdb70 database. An initial evaluation based on AlphaFold’s PAE score indicated that the position of the first ~285 amino acids relative to the rest of the protein could not be determined at sufficient predicted accuracy. Thus, we divided the protein sequence into N- and C-terminal parts, consisting of residues 1 through 285, and 286 through 1254, respectively, and used AlphaFold to construct model structures of each part separately. Next, we performed a ConSurf evolutionary conservation analysis [
18] with the two models. Homologs were collected using HMMER [
19] search against the UniRef90 database with E-values of 1 × 10
−5 and sequence identity ranging from 35% to 95%. Sixty-five homologs were detected for the N-terminal part, and five hundred for the C-terminal part. All detected homologs were of fungal origin. Thus, to identify sequence regions that are unique to fungi and are not found in other eukaryotes, a more thorough homologue search was conducted. Specifically, homologs were searched and aligned using HMMER against all eukaryotic genomes in the UniProt representative proteomes database for both the N- and C-terminal domains, with a 15% co-membership threshold (RP15), an E-value of 1 × 10
−5, and no constraints on sequence identity. Using that search, 1713 homologs were collected for the N-terminal domain, and 9510 homologs for the C-terminal domain. The RP15 database was chosen to reduce the number of sequences for the initial search. Finally, regions that emerged as potential fungal-specific regions were subjected to a new HMMER search against the full UniProt KB database.
Microscopy. CrpA_dN, CrpA_dmid, and CrpA_cont strains were grown on coverslips in MMVUU without copper in 24-well plates for 16 h. The CrpA_cont strain was exposed to different concentrations of copper (0.25, 2.5, 25, and 100 µM) for various time exposures after 16 h of growth. In subsequent experiments, CrpA_dN, CrpA_dmid, and CrpA_cont strains were exposed to 2.5 µM Cu for 2 h. The coverslips were then washed twice with DDW and treated accordingly. Co-localization of the GFP-tagged CrpA protein with the endoplasmic reticulum (ER) or nuclei, was performed with ER-Tracker Red (Invitrogen) or DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich, St. Louis, MO, USA), respectively. Nuclear staining was carried out for 20 min at room temperature using DAPI solution (50 mM KPO4 pH 6.8, 0.2% (v/v) Triton X-100, 5% (v/v) glutaraldehyde, 0.05 µg/mL DAPI). ER staining was performed with 1 µM ER-Tracker Red for 15 min at 37 °C. Microscopy was performed on a Zeiss LSM 800 confocal laser scanning microscope (Zeiss, Jena, Germany).
Droplet-growth-inhibition assay. Freshly harvested conidia were serially diluted in sterile water to obtain defined concentrations of 106, 105, 104, and 103 conidia/mL. Conidia were spotted in a volume of 10 µL on MMVUU plates in the presence of increasing concentrations of Cu. Growth was documented after 72 h of incubation at 37 °C.
Liquid broth inhibition assay. Freshly harvested conidia (5000 conidia/well), suspended in 200 µL/well MMVUU liquid medium were dispensed in 96-well plates, with increasing concentrations of Cu. The minimal inhibitory concentration (MIC), namely the Cu concentration in which no microscopic growth was visible, was measured after 24 h of incubation at 37 °C.
Mouse alveolar macrophage conidial killing assay. Mouse alveolar macrophages were collected from 8-week-old ICR mice as previously described [
20]. Conidia (2 × 10
5) were added to freshly harvested murine alveolar macrophages in 24-well plates at a 1:1 ratio for 2 h of preincubation at 37 °C under 5% CO
2 in DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin medium. Uninternalized conidia were aspirated and the wells were washed with PBS three times. Then, 1 mL RPMI medium was added for 4 h of incubation at 37 °C and 5% CO
2. Conidial killing was terminated by aspiration of the RPMI medium and addition of DDW with 0.1% Triton X-100 to lyse the macrophages. Following vigorous scraping of the wells, lysate dilutions were plated on YAG agar plates with 0.01% chloramphenicol and incubated for 24 h at 37 °C to detect viable fungal colonies. Viable counts were compared to those of conidia incubated with macrophages at the 0 h time point (after the 2 h preincubation). The results were analyzed using the Brown–Forsythe ANOVA test with Dunnett’s T3 multiple comparisons post-test in GraphPad Prism software.
Mouse model of A. fumigatus infection. A standard model of mouse infection was used [
21]. Six-week-old ICR female mice were injected subcutaneously with 300 mg/kg cortisone–acetate 3 days before infection, on the day of infection, and 2 and 4 days post-infection to induce an immunocompromised state but without neutropenia. The mice were infected intranasally with 5 × 10
5 dormant conidia/mouse, suspended in 20 µL of 0.2% Tween 20 in saline (10 µL in each nostril). Survival was monitored for 14 days. The results were analyzed using the log-rank test for Kaplan–Meyer survival curves in GraphPad Prism software.
Statistical analysis. Data and statistical analysis were analyzed using GraphPad Prism 5 software package (GraphPad Software, Inc., San Diego, CA, USA) or Microsoft Excel software package (Microsoft Corporation, Redmond, WA, USA). Student’s t-test was used for significance testing of two groups. Differences between the groups were considered significant at p ≤ 0.05.
4. Discussion
Previous work reported two critical proteins in
A. fumigatus resistance to toxic Cu levels mounted by the host defense system, namely, the Cu-homeostasis transcription factor AceA and the Cu-exporting ATPase CrpA, positively regulated by AceA [
6,
9,
11]. Deletion of
A. fumigatus crpA results in Cu hypersensitivity, reduced survival in the presence of mouse alveolar macrophages, and significantly decreased virulence [
9]. We therefore decided to investigate the function of CrpA in more detail. In particular, we sought to identify fungal-specific motifs necessary for CrpA function that could be considered as drug targets.
We constructed a model structure of CrpA in silico using AlphaFold and identified two fungal-unique sequences. The first is an N-terminal cytosolic tail (amino acids 1–211), found only in the filamentous ascomycetes. Within it, a ~110 amino acid section spanning roughly between positions 90 and 200 can be found only in the genus Aspergillus. The second is an intracellular loop containing a unique conserved motif (amino acids 542–556) found only in fungi and not in other eukaryotic organisms.
We generated
A. fumigatus strains in which the N-terminal cytosolic tail was deleted (CrpA_dN) or the intracellular loop replaced with a flexible glycine–serine linker (CrpA_dmid) [
22]. Deleting the N-terminal cytosolic tail resulted in a moderate increase in Cu sensitivity, and localization of the protein was the same as of wild-type CrpA. There are five predicted Cu-binding domains at the N-terminus of
A. fumigatus CrpA, and two are located closer to the N-terminus and shared by
A. nidulans and
C. albicans, followed by three that are conserved in other fungi [
14]. The CrpA_dN strain we generated lacks the two Cu-binding domains closest to the N-terminus (CxxC), and its moderate sensitivity to excess Cu can be explained by the existence of the three remaining metal-binding domains located downstream of the part we deleted. We infer that these three metal-binding domains can bind Cu efficiently enough for the CrpA protein to function at an intermediate level compared to that of the control strain.
In contrast, deletion of the CrpA intracellular loop resulted in high Cu sensitivity. Furthermore, CrpA_dmid was concentrated in the ER and unlike normal CrpA, was not observed at the cell surface. This finding indicates that the fungal-specific intracellular loop (CrpA amino acids 542–556) is essential for trafficking or localizing CrpA to the cell surface. Failure to localize there leads to an inability to efflux excess Cu, and to Cu sensitivity only slightly less severe than that of the
crpA null strain. On the basis of these findings, the intracellular loop could (i) serve as a motif that directly anchors CrpA to the cell surface, consistent with the high occurrence of arginine residues and aromatic amino acids, known to interact favorably with membrane-lipid head-groups [
23]. Alternatively, (ii) the intracellular loop could serve as a motif that targets CrpA for loading into vesicles at the trans-Golgi network and transport via the secretory SEC pathway to the cell surface.
Surprisingly, we detected that although CrpA_dmid was almost as sensitive as the CrpA deletion strain to excess Cu in vitro, unlike the latter, it displayed wild-type virulence in infected mice. This finding suggests that even the small increase in protection against Cu seen in CrpA_dmid compared to ΔCrpA, is sufficient to significantly protect it against Cu stress after ingestion by macrophages and neutrophils. This rationale is strengthened by our results showing that internalized conidia of CrpA_dmid are significantly more resistant to killing by mouse alveolar macrophages compared to ΔCrpA (p < 0.0001). Importantly, in our infection model, mouse alveolar macrophages are weakened due to cortisone–acetate administration, which is essential for disease progression. As a result, even the residual Cu resistance in CrpA_dmid could be sufficient to overcome the Cu stress induced by these compromised immune cells.
Collectively, the findings described here and in our previous work [
9] show that antifungal therapy based solely on inhibiting CrpA Cu efflux, will, for several reasons, be only partially effective. First, CrpA deletion, attenuates but does not completely block
A. fumigatus virulence, and second, even residual CrpA activity, as in the CrpA_dmid strain, results in wild-type virulence. Therefore, if fungal-specific CrpA-dependent Cu-efflux inhibitors are developed, they will have to be used with additional potentiating or synergistic drugs.