Abstract:

:Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, is considered as an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-CoA acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported critical for cell viability. Using yeast ERG10 for alignment, two orthologues AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570) were discovered in opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B has been previously validated, the biological function of AfERG10A remains unclear. In this study, we have characterized recombinant AfERG10A as a functional acetyl-CoA acetyltransferase catalyzing both synthetic and degradative reactions. Unexpectedly, AfERG10A localizes to mitochondria in A. fumigatus as shown by C-terminal GFP-tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus Reduced expression of Aferg10A leads to severe morphological defects and increased susceptibility to oxidative and cell wall stresses. Although the catalytic mechanism of acetyl-CoA acetyltransferase family is highly conserved, the crystal structure of AfERG10A and its complex with CoA are solved revealing four substitutions within the CoA binding site that are different from human orthologues. Taken together, our combination of genetic and structural studies demonstrates that mitochondrial AfERG10A is essential for A. fumigatus cell viability and could be a potential drug target to feed antifungal drug development pipeline.Importance A growing number of people worldwide are suffering from invasive aspergillosis, caused by human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on limited repertoire of antifungals. Ergosterol is an essential component of the fungal cell membrane as well as a target of current antifungals. Approximately 20 enzymes are involved in ergosterol biosynthesis of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. Two ACATs in A. fumigatus are AfErg10A and AfErg10B. However, the biological function of AfErg10A is yet to be investigated. In this study, we showed that AfErg10A is localized in the mitochondria, and is essential for A. fumigatus survival and morphological development. In combination with structural studies we validated AfErg10A as a potential drug target that will facilitate the development of novel antifungals and improve the efficiency of existing drugs.