The Role of Zinc in Copper Homeostasis of Aspergillus fumigatus

Abstract

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.

Keywords: Aspergillus fumigatus; CtrC; ZafA; copper; zinc.

Figure 1

Zinc starvation suppressed the growth defect of the AfMac1 deletion mutant. To investigate the effect of zinc on growth of the AfMac1 deletion mutant, a spotting assay was performed with the wild-type, Δafmac1, and AfMac1 complemented strains of A. fumigatus. (A) Conidia (10 × 10⁵) of the indicated strain were spotted on AMM (77 mM zinc), zinc-starved (1 µM zinc) medium, copper-supplemented (50 mM copper) medium, or BCS-supplemented medium and incubated for 48 h at 37 °C. (B) Additionally, CtrC was introduced into the Δafmac1 strain, and the effect of CtrC was investigated. Conidia (10 × 10⁵) of the indicated strain were spotted on AMM or zinc-starved (1 µM zinc) medium and incubated for 48 h at 37 °C.

Figure 1

Zinc starvation suppressed the growth defect of the AfMac1 deletion mutant. To investigate the effect of zinc on growth of the AfMac1 deletion mutant, a spotting assay was performed with the wild-type, Δafmac1, and AfMac1 complemented strains of A. fumigatus. (A) Conidia (10 × 10⁵) of the indicated strain were spotted on AMM (77 mM zinc), zinc-starved (1 µM zinc) medium, copper-supplemented (50 mM copper) medium, or BCS-supplemented medium and incubated for 48 h at 37 °C. (B) Additionally, CtrC was introduced into the Δafmac1 strain, and the effect of CtrC was investigated. Conidia (10 × 10⁵) of the indicated strain were spotted on AMM or zinc-starved (1 µM zinc) medium and incubated for 48 h at 37 °C.

Figure 2

CtrC expression was regulated by zinc and ZafA. To investigate the effect of zinc on the expression of Ctr genes, Northern blotting was performed. (A) Cells were cultured in different media containing the indicated metal concentrations until mid-log phase. Total RNA was extracted, and then Northern blotting was performed (77 µM zinc and 6.4 µM copper indicate AMM, 0 µM zinc indicates zinc starvation medium, 500 µM zinc indicates zinc sufficient medium, and 50 µM copper indicates copper sufficient medium). The probes targeted CtrA1, CtrA2, CtrB, and CtrC, which are members of the Ctr gene family. (B) The effect of different transcription factors involved in metabolism of diverse metals on the expression CtrC was investigated. The deletion mutant of the indicated gene was cultured in AMM, and total RNA was extracted. HapX is an iron-regulated transcriptional activator. (C) The effect of zinc on the expression of CtrC was evaluated in the Δafmac1 strain. The wild-type and Δafmac1 strains were cultured in the indicated zinc concentration until mid-log phase, and total RNA was extracted (1 µM zinc indicates zinc starvation media, and 77 µM zinc indicates AMM). (D) Sequencing analysis of the promoter region of the Ctr gene family was performed. The conserved ZafA binding motif 5′-CAA(G)GGT-3′ was found in the promoter regions of CtrA1, CtrA2, and CtrC but not in that of CtrB.

Figure 3

ZafA directly binds to the promoter region of CtrC. The conserved ZafA binding motif 5′-CAA(G)GGT-3′ was found in the promoter regions of CtrA1, CtrA2, and CtrC. To investigate the role of ZafA in the expression of Ctr genes, an EMSA experiment was performed with DNA fragments that contained the conserved ZafA binding motif. Briefly, 118, 114, and 128 bp DNA fragments of the promoter regions of CtrA1, CtrA2, and CtrC, respectively, which contain the conserved ZafA binding motif, were amplified. P³²-labeled probes (hot) were reacted with recombinant ZafA protein, and the reaction mixtures were separated via PAGE. Unlabeled probes (cold) were used as a competitor.

Figure 4

The ZafA binding motif has an important role in CtrC transcription induced by ZafA under zinc starvation conditions. To identify the role of the ZafA binding motif localized in the CtrC promoter region, β-galactosidase assays and Northern blotting were performed with a mutant ZafA binding motif. (A) The β-galactosidase assay was performed with a strain coexpressing ZafA and CtrC. CtrC and ZafA genes of A. fumigatus were subcloned into a yeast plasmid that encodes the LacZ reporter gene, and the yeast BY4741 strain was cotransformed with both plasmids. EV indicates empty vector, and CtrC-M indicates the mutant ZafA binding motif of the CtrC promoter region (Figure S1). Then, β-galactosidase activity was measured as described in materials and methods (p < 0.0001). (B) The expression of CtrC when the ZafA binding motif was mutated was investigated. A mutant strain of the ZafA binding motif of CtrC was constructed from wild-type and Δafmac1 strains (Figure S2), and the indicated strains were cultured in media with different zinc concentrations (1 and 77 µM zinc). Northern blotting was performed to investigate the expression of CtrC.

Figure 5

Mutation of the ZafA binding motif of CtrC led to failure of ZafA binding. To identify the binding affinity of ZafA to the ZafA binding motif of CtrC, an EMSA experiment was performed with a mutant ZafA binding motif of CtrC. (A) The recombinant ZafA strongly bound to the 95 bp length of the ZafA promoter region, and a cold probe acted as a competitor. (B) The binding affinity of ZafA to the mutant ZafA binding motif of CtrC was investigated. The cold wild-type ZafA binding motif and mutant form of the ZafA binding motif were used as competitors in the EMSA experiment.

Figure 5

Mutation of the ZafA binding motif of CtrC led to failure of ZafA binding. To identify the binding affinity of ZafA to the ZafA binding motif of CtrC, an EMSA experiment was performed with a mutant ZafA binding motif of CtrC. (A) The recombinant ZafA strongly bound to the 95 bp length of the ZafA promoter region, and a cold probe acted as a competitor. (B) The binding affinity of ZafA to the mutant ZafA binding motif of CtrC was investigated. The cold wild-type ZafA binding motif and mutant form of the ZafA binding motif were used as competitors in the EMSA experiment.

Figure 6

The binding affinity of ZafA to the ZafA binding motif of CtrC was inversely proportional to the copper or zinc concentration. To investigate the effect of zinc and copper on the binding affinity of ZafA to the ZafA binding motif of CtrC, the indicated zinc or copper concentration was used in the reaction mixture, and an EMSA experiment was performed.