SAGA Complex Subunit Hfi1 Is Important in the Stress Response and Pathogenesis of Cryptococcus neoformans.

The Spt-Ada-Gcn Acetyltransferase (SAGA) complex is a highly conserved co-activator found across eukaryotes. It is composed of a number of modules which can vary between species, but all contain the core module. Hfi1 (known as TADA1 in Homo sapiens) is one of the proteins that forms the core module, and has been shown to play an important role in maintaining complex structural integrity in both brewer's yeast and humans. In this study we successfully identified the gene encoding this protein in the important fungal pathogen, Cryptococcus neoformans, and named it HFI1. The hfi1Δ mutant is highly pleiotropic in vitro, influencing phenotypes, ranging from temperature sensitivity and melanin production to caffeine resistance and titan cell morphogenesis. In the absence of Hfi1, the transcription of several other SAGA genes is impacted, as is the acetylation and deubiquination of several histone residues. Importantly, loss of the gene significantly impacts virulence in a murine inhalation model of cryptococcosis. In summary, we have established that Hfi1 modulates multiple pathways that directly affect virulence and survival in C. neoformans, and provided deeper insight into the importance of the non-enzymatic components of the SAGA complex.

amdS as a dominant recyclable marker in Cryptococcus neoformans.

While the fungal pathogen Cryptoccocus neoformans is a leading cause of death in immunocompromised individuals, the molecular toolkit currently available to study this important pathogen is extremely limited. To enable an unprecedented level of control over manipulation of the genome, we have developed a dominant recyclable marker by expanding on the classic studies of the amdS gene by Michael J. Hynes and John Pateman. The ascomycete Aspergillus nidulans employs the acetamidase AmdS to hydrolyse acetamide to ammonium and acetate, which serve as a nitrogen and carbon source, respectively. Acetamidase activity has never been reported in the Basidiomycota. Here we have successfully demonstrated that acetamide can be utilized as a good nitrogen source in C. neoformans heterologously expressing amdS and that this activity does not influence virulence, enabling it to be used as a basic dominant selectable marker. The expression of this gene in C. neoformans also causes sensitivity to fluoroacetamide, permitting counterselection. Taking advantage of this toxicity we have modified our basic marker to create a comprehensive series of powerful and reliable tools to successfully delete multiple genes in the one strain, generate markerless strains with modifications such as fluorescent protein fusions at native genomic loci, and establish whether a gene is essential in C. neoformans.