Abstract
Copper (Cu) is an essential metal for all living organisms, although it is toxic in excess. Filamentous fungus must acquire copper from its environment for growth. Despite its essentiality for growth, the mechanisms that maintain copper homeostasis are not fully understood in filamentous fungus. To gain insights into copper homeostasis, we investigated the roles of a copper transcription factor Afmac1 in the life-threatening fungus Aspergillus fumigatus, a homolog of the yeast MAC1. We observed that the Afmac1 deletion mutant exhibited not only significantly slower growth, but also incomplete conidiation including a short chain of conidia and defective melanin. Moreover, the expressions of the copper transporters, ctrA1, ctrA2, and ctrC, and metalloreductases, Afu8g01310 and fre7, were repressed in ∆Afmac1 cells, while those expressions were induced under copper depletion conditions in wild-type. The expressions of pksP and wetA, which are, respectively, involved in biosynthesis of conidia-specific melanin and the late stage of conidiogenesis, were decreased in the ∆Afmac1 strain under minimal media condition. Taken together, these results indicate that copper acquisition through AfMac1 functions in growth as well as conidiation.
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Acknowledgements
We would like to thank Taku Oshima (Nara Institute of Science and Technology) and Takashi Umeyama (National Institute of Infectious Diseases) for their fruitful discussions, and Ryoko Mori for experimental assistance. This work has been partly supported by the Takeda Science Foundation to H. T., MEXT KAKENHI (Nos. 16K18671, 16H06279 to H. T., and 25290079 to T. Y.), the Institute for Global Prominent Research, Chiba University to H.T., and the Tenure Tracking System Program of MEXT to Y.K. and H.T. We thank the National BioResource Project—Pathogenic Microbes in Japan (http://www.nbrp.jp/).
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Communicated by M. Kupiec.
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294_2017_681_MOESM1_ESM.pdf
Split-marker strategy for the ∆Afmac1 strain. (a) The preparation of two fusion PCR products. Fragments A and B were amplified from A. fumigatus chromosomes. The figure is a schematic representation of the location of primers used for recombinant PCR. The ptrA gene was amplified from the pPTRI plasmid. (b) Homologous recombination. The two fusion PCR products were used directly for transformation. Homologous recombination between the overlapping regions of ptrA, and between the flank regions and chromosomal DNA results in Afmac1 deletion (PDF 155 KB)
294_2017_681_MOESM2_ESM.pdf
Confirmation of the constructed mutants, ∆Afmac1 and Co-Afmac1. (a) Genomic architectures of Afmac1::ptrA in ∆Afmac1 and Afmac1-HygB rin Co-Afmac1. (b) Agarose gel electrophoresis of PCR products. Five primer sets were designed for confirmation of ∆Afmac1 and Co-Afmac1. (c) Quantitative real-time RT-PCR analyses of Afmac1 in WT, ∆Afmac1, and Co-Afmac1 strains. Average values and error bars were obtained from three experiments (PDF 240 KB)
294_2017_681_MOESM4_ESM.pdf
Phialides and conidia of WT and ∆Afmac1 strain. The structure of conidiogenesis was observed by performing a slide culture on the AMM medium (PDF 569 KB)
294_2017_681_MOESM5_ESM.pdf
Quantitative real-time RT-PCR analyses of wetA and pksP at a high copper concentration (AMM containing 100 µM CuSO4). Average values and error bars were obtained from three independent experiments (PDF 138 KB)
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Kusuya, Y., Hagiwara, D., Sakai, K. et al. Transcription factor Afmac1 controls copper import machinery in Aspergillus fumigatus . Curr Genet 63, 777–789 (2017). https://doi.org/10.1007/s00294-017-0681-z
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DOI: https://doi.org/10.1007/s00294-017-0681-z