Aspergillus fumigatus Mitochondrial Acetyl Coenzyme A Acetyltransferase as an Antifungal Target.

Yuanwei Zhang,Ling Lu,Jialu Fan,Wenxia Fang,Cheng Jin,Wenfan Wei,Claire Vieille

doi:10.1128/aem.02986-19

Abstract

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. In yeast, the enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. Using yeast ERG10 for alignment, two orthologues, AfERG10A (AFUB_000550) and AfERG10B (AFUB_083570), were discovered in the opportunistic fungal pathogen Aspergillus fumigatus Despite the essentiality of AfERG10B having 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 the mitochondria in A. fumigatus, as shown by C-terminal green fluorescent protein (GFP) tag fusion. Both knockout and inducible promoter strategies demonstrate that Aferg10A is essential for the survival of A. fumigatus The 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 the antifungal drug development pipeline.IMPORTANCE A growing number of people worldwide are suffering from invasive aspergillosis caused by the human opportunistic fungal pathogen A. fumigatus Current therapeutic options rely on a 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.

Highlights

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy
The first step is synthesized by acetyl-coenzyme A (CoA) acetyltransferase (ACAT) converting two acetyl-CoA into acetoacetyl-CoA, which is subsequently converted by 3-hydroxy-3-methyl-glutaryl (HMG)-CoA synthase and HMG-CoA reductase to mevalonate as the precursor for ergosterol biosynthesis [9]
The binding affinities for acetoacetyl-CoA and CoA are higher than that for acetyl-CoA (Table 1). These data suggest that Aferg10A (AFUB_000550) encodes an active acetyl-CoA acetyltransferase in A. fumigatus

Summary

Introduction

Ergosterol plays an important role in maintaining cell membrane sterol homeostasis in fungi, and as such, it is considered an effective target in antifungal chemotherapy. The enzyme acetyl-coenzyme A (CoA) acetyltransferase (ERG10) catalyzes the Claisen condensation of two acetyl-CoA molecules to acetoacetyl-CoA in the ergosterol biosynthesis pathway and is reported as being critical for cell viability. 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 the antifungal drug development pipeline. 20 enzymes are involved in ergosterol biosynthesis, of which acetyl-CoA acetyltransferase (ACAT) is the first enzyme. In Arabidopsis thaliana, ACAT1 and ACAT2 have been identified, and only ACAT2 is essential and required for normal growth and development [18] Both are able to rescue the lethal phenotype of the S. cerevisiae erg mutant, indicating that the function of ACAT is highly conserved [18]. Each monomer consists of three domains, the N-domain and the C-domain, which share the same ␤␣␤␣␤␣␤␤ topology, and the loop domain, which is involved in tetrameric assembly and provides binding pocket for the CoA moiety [22]

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Results

Conclusion