WHO lists the environmental yeast Cryptococcus neoformans as a critical priority pathogen, with an estimated 180,000 global deaths a year. The progression of the disease from a pulmonary infection to lethal meningoencephalitis is dependent on multiple metabolic and structural features classified as virulence traits. Regulating these virulence traits is dependent on transcriptional regulators. One of the regulators functioning through histone modification is called the Spt-Ada-Gcn Acetyltransferase (SAGA) complex.
Previous studies into C. neoformans SAGA were focused primarily on individual candidate proteins predicted to be part of the complex. The deletion of these protein-coding genes leads to several unique virulence phenotypes. Furthermore, loss-of-function mutation of candidate SAGA subunit Sgf29 was identified within clinical isolates, with the gene-deletion strain generated in our laboratory, it is showed to be hypervirulent. Such microevolution also exists in other SAGA candidate subunits based on bioinformatic analyses. Therefore, the mutations within SAGA may explain some isolates switch from an environmental to a pathogenic species. It is an intriguing target to investigate C. neoformans pathogenesis and may provide awareness of further treatment. However, within fungi, the conservation of these proteins as a cohesive complex has not been demonstrated outside of the Asomycota branch, and only phenotypic assay linking these proteins within C. neoformans.
To study proteomic interactions and determine the composition of this complex, one of the ways is through tandem affinity purification, which is a gentle and robust technique. Here, we developed a modified protocol based of tandem affinity purification, which includes a codon-optimised dual tag affinity integrated with the recyclable marker system.