Molecular Characterization and the Essential Biological Function of the Metal Chaperone Protein MtmA in Aspergillus fumigatus

Abstract

The detoxification system of reactive oxygen species (ROS) plays critical roles in the survival and virulence of fungal pathogens in infected hosts, while superoxide dismutase (SOD) is the primary ROS scavenger. In the model yeast Saccharomyces cerevisiae, the metal chaperone protein Mtm1 is required for mitochondrial Sod2 activation and responses to oxidative stress. However, the function of the S. cerevisiae Mtm1 homolog in the human fungal pathogen Aspergillus fumigatus has not yet been clarified. In this study, we found that mitochondria-localized MtmA in A. fumigatus, a putative homolog of yeast Mtm1, not only has a similar function to Mtm1 in responding to oxidative stress resistance by affecting SodB (MnSOD) activity but is also essential for hyphal growth such that repressed expression of MtmA results in severe growth defects in A. fumigatus. In addition, the chelation of Zn²⁺ can obviously rescue growth defects caused by repression of MtmA, suggesting that MtmA may be involved in hyphal growth by affecting cellular Zn²⁺ detoxification. Moreover, MtmA contains four Mito-carr domains, whereas only the first Mito-carr domain is required for the function of MtmA. Therefore, the findings in this study suggest that MtmA in A. fumigatus has an important and unique function that is different from that in yeast. IMPORTANCE Knowledge of the key factors required for the viability of pathogenic fungi can help to explore new antifungal drugs. Here, we demonstrate that MtmA is involved in responding to oxidative stress by activating mitochondrial SodB activity. MtmA, especially for the first Mito-carr domain, is essential for colony growth by regulating cellular Zn²⁺ equilibrium and responses to oxidative stress in A. fumigatus. This is the first report of the vital and unique role of the MtmA protein in pathogenic fungi, indicating that it might be a potential antifungal drug target.

Keywords: Aspergillus fumigatus; ROS; oxidative stress; superoxide dismutase.

FIG 1

MtmA is localized to the mitochondria in A. fumigatus. (A) Diagram illustrating the strategy for construction of the MtmA::GFP strain under the control of a native promoter. (B) Colony morphologies of the indicated strains grown on YAG medium and MM for 2 days at 37°C. (C) MtmA::GFP is colocalized with mitochondria-localized MrsA::RFP. Scale bar, 10 μm.

FIG 2

The essential gene mtmA in A. fumigatus was identified and analyzed using the heterokaryon rescue technique. (A) Diagram illustrating the deletion strategy for mtmA. (B) Diagnostic PCR showed that WT only contains the mtmA gene allele (1,615 bp, primers mtmA-S and mtmA-A) and that the heterokaryon contains both the mtmA gene and deletion alleles of the integrated pyr4 nutritional marker (1,933 bp, primers mtmA-P1/pyr4-up). (C) Conidia from four primary transformants by mtmA deletion cassette transformation were streaked on selective (YAG) and nonselective (YUU) plates for 48 h at 37°C. (D) Conidial spores from heterokaryon transformants and WT cultured in liquid YAG for 24 h, showing that the conidial spores were nongerminated. (E) Phenotypes of abnormal germlings in progeny of mtmA deletion transformants.

FIG 3

Growth phenotypes of the P_alcA::mtmA and P_niiA::mtmA conditional strains under inducing and repressing growth conditions. (A) Colony phenotype comparison of P_niiA::mtmA and A1160 cultured on the inducing medium MMUU at 37°C for 3 days and the repressing medium MMUU(NH₄⁺) at 37°C for 3 or 4 days. (B) Comparison of the colony phenotype of P_alcA::mtmA in inducing and repressing medium 48 h at 37°C. (C) Comparison of the growth phenotype of P_alcA::mtmA in liquid-inducing and -repressing medium for 12 h at 37°C. (D) qRT-PCR analysis of the mRNA expression levels of the mtmA gene under inducing and repressing conditions. P values were calculated using Student's t test with Welch’s correction: ***, P < 0.001. (E) Accumulated rhodamine 123 in strains detected by flow cytometry showing the mitochondrial membrane potential. FL1-A on the x axis represents the relative fluorescence intensity value.

FIG 4

Overexpression of sodB could restore the oxidative stress sensitivity induced by the repression of MtmA. (A) Conidia of each indicated strain were inoculated on repression (YAG) or induction (MM plus glycerol) medium containing serial concentrations of menadione and H₂O₂ for 1.5 or 2 days at 37°C. (B) Mycelium growth inhibition ratios of the indicated strains on YAG and MM plus glycerol with different concentrations of menadione and H₂O₂. *, P < 0.05; ***, P < 0.001; ns, not significant. (C) Colony morphologies of the indicated strains grown on YAG medium containing a serial concentration of menadione for 1.5 days at 37°C. (D) Mycelium growth inhibition ratios of the indicated strains on YAG with different concentrations of menadione. Different letters imply significant differences between the samples (P < 0.05, Duncan’s test). (E) Measurement of the activity of SodA and SodB in the indicated strains by nitroblue tetrazolium (NBT) staining. Actin was used as the loading control. The value of integrated optical density (IOD; IOD_{target protein}/IOD_actin) measured by ImageJ shows the relative protein quantity. (F) Western blot analysis of MtmA::GFP under treatment with 1 mM Mn²⁺ for 1 and 2 h. Actin was used as the loading control. The value of the integrated optical density (IOD; IOD_{target protein}/IOD_actin) measured by ImageJ shows the relative protein quantity.

FIG 5

EDTA could significantly restore the growth defects caused by the repression of MtmA but not oxidative stress sensitivity. (A) Colony morphologies of the indicated strains grown on YAG medium containing a serial concentration of EDTA for 2 days at 37°C. (B) Hyphal morphologies of the indicated strains grown on YAG in the presence or absence of 0.5 mM EDTA for 12 h at 37°C. Scale bar, 10 μm. (C) qRT-PCR analysis of the mRNA expression levels of the mtmA gene under EDTA treatment. The P value was calculated by Student's t test: ***, P < 0.001. (D) Phenotypic characterization of the indicated strains with or without the addition of EDTA (1 mM) in the presence of menadione (10 and 15 μM). (E) SodA and SodB activities of related strains were measured when cultured with EDTA. Actin was used as the loading control. (F) SodB activity of P_alcA::mtmA was normalized to that of the parental wild-type strain (wild type). ***, P < 0.001.

FIG 6

The chelation of Zn²⁺ could rescue growth defects caused by the repression of MtmA. (A) Colony morphologies of the indicated strains grown on YAG medium in the presence of 1.5 mM EDTA at 37°C for 2 days (with MnCl₂, ZnCl₂, FeSO₄, CuCl₂, CaCl₂, MgCl₂, and CoCl₂). (B) Phenotypic characterizations of the indicated strains with or without the addition of EDTA (1.5 mM) in the presence of Zn²⁺ (0.125, 0.25, 0.5 mM). (C) Colony morphologies of the indicated strains grown on YAG medium in the presence or absence of serial concentrations of TPEN at 37°C for 2 days. (D) Hyphal morphologies of the indicated strains grown on YAG in the presence or absence of 100 μM TPEN at 37°C for 12 h or 14 h. Scale bar, 10 μm.

FIG 7

The first Mito-carr domain of MtmA is required for the growth and oxidative stress resistance of A. fumigatus. (A) Schematic view of the serial Mito-carr domain truncation in MtmA. ΔN, deletion of the region from amino acids 2 to 55 (aa 2 to 55); Δ1, deletion of Mito-carr domain 1 (aa 56 to 103); Δ2, deletion of Mito-carr domains 1 and 2 (aa 125 to 174); ΔC, deletion of Mito-carr domains 3 and 4 (aa 257 to 464). (B) qRT-PCR analysis was performed after cultures were grown in MM for 24 h at 37°C. The tubA gene was used as an internal control. The P value was calculated by Student's t test: ***, P < 0.001; ns, P > 0.05 compared to the parental wild-type strain. (C) Colony morphologies of the indicated strains grown on YAG medium in the presence or absence of 10 or 15 μM menadione at 37°C for 36 h.