Tools for genetic manipulation of the endemic fungal pathogen <i>Emergomyces africanus</i> and application of a fluorescent reporter strain in infection models

ABSTRACT Emergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa, which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterization of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of Histoplasma capsulatum were adapted for use in E. africanus. Agrobacterium -mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall α(1,3)-glucan by targeting the transcripts of the α-glucan synthase ( AGS1 ). An episomal CRISPR/Cas9 system was evaluated for E. africanus , which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus . IMPORTANCE Emergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognized in recent years, and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (i) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (ii) methods for knocking down gene expression in E. africanus , and (iii) the permanent disruption of genes through CRISPR/Cas9 gene editing.