Abstract
Dihydroxyacid dehydratase (DHAD) is a key enzyme in the branched-chain amino acid biosynthetic pathway that exists in a variety of organisms, including fungi, plants and bacteria, but not humans. In this study we identified four putative DHAD genes from the filamentous fungus Aspergillus fumigatus by homology to Saccharomyces cerevisiae ILV3. Two of these genes, AFUA_2G14210 and AFUA_1G03550, initially designated AfIlv3A and AfIlv3B for this study, clustered in the same group as S. cerevisiae ILV3 following phylogenetic analysis. To investigate the functions of these genes, AfIlv3A and AfIlv3B were knocked out in A. fumigatus. Deletion of AfIlv3B gave no apparent phenotype whereas the Δilv3A strain required supplementation with isoleucine and valine for growth. Thus, AfIlv3A is required for branched-chain amino acid synthesis in A. fumigatus. A recombinant AfIlv3A protein derived from AFUA_2G14210 was shown to have DHAD activity in an in vitro assay, confirming that AfIlv3A is a DHAD. In addition we show that mutants lacking AfIlv3A and ilv3B exhibit reduced levels of virulence in murine infection models, emphasising the importance of branched-chain amino acid biosynthesis in fungal infections, and hence the potential of targeting this pathway with antifungal agents. Here we propose that AfIlv3A/AFUA_2G2410 be named ilvC.
Highlights
Aspergillus fumigatus is a filamentous fungus and an opportunistic human pathogen, which primarily affects immunocompromised patients
Saccharomyces cerevisiae ilv2D has greatly decreased ability to grow in vivo [7], and virulence is decreased when ILV2 is disrupted in Cryptococcus neoformans [8] and Candida albicans [9]
As the relationship of these proteins to ILV3 was not clear, a phylogenetic analysis was undertaken using in addition sequences homologous to ILV3 from other fungi, bacteria and archeabacteria
Summary
Aspergillus fumigatus is a filamentous fungus and an opportunistic human pathogen, which primarily affects immunocompromised patients. The numbers of patients with invasive fungal infections has risen dramatically over the last 20 years [1,2]. Certain fungal biosynthetic pathways provide attractive targets for antifungal research because they are absent in mammals One such pathway is the branched-chain amino acid biosynthetic pathway, which synthesises isoleucine, leucine and valine in a range of organisms from bacteria to eukaryotes, including fungi and higher plants [4]. This pathway is the target of several different herbicides that inhibit the first common enzyme in the pathway, acetolactate ( known as acetohydroxyacid) synthase [5,6]. Saccharomyces cerevisiae ilv2D has greatly decreased ability to grow in vivo [7], and virulence is decreased when ILV2 is disrupted in Cryptococcus neoformans [8] and Candida albicans [9]
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