An ancient gene duplication is implicated in virulence in the human pathogen, Histoplasma

ABSTRACT Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the α-amylase Amy1p, as essential for α-glucan production and virulence in Histoplasma capsulatum. We identified two new genes (AMY2 and AMY3) in the Histoplasma genome that encode putative α-amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in α-glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3, and establishing how many AMY copies existed across different fungi. We found that the number and type of α-amylases vary depending on the fungal species; that all α-amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasma’s Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma α-glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for α-glucan biosynthesis and virulence. Competing Interest Statement The authors have declared no competing interest.