Coordinate genomic association of transcription factors controlled by an imported quorum sensing peptide in Cryptococcus neoformans

Abstract

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression.

The Transcription Factor Pdr802 Regulates Titan Cell Formation and Pathogenicity of Cryptococcus neoformans

“…Of these, 540 occurred within 1,000 bp upstream of transcription start sites ( Data Set S1 , sheets 1 and 2), which we used as an approximation of regulatory regions. Notably, ChIP-Seq data for the region upstream of the PDR802 transcription start site suggested self-regulation, as has been reported for other cryptococcal TFs ( 74 , 75 ) ( Fig. S7B ).…”

“…Our analysis showed that Pdr802 positively regulates expression of LIV3 and STB4 , while it negatively regulates ZFC3 ( Table 1 ), in concordance with our phenotypic observations of Titan cell formation. Notably, Liv3 and Zfc3 are responsive to the peptide Qsp1 ( 74 , 75 ) and are important for C. neoformans virulence, while Stb4 influences cryptococcal brain infection ( 67 ).…”

“…We have further identified multiple genes that are central in these processes and are directly regulated by Pdr802. Some of these targets are also regulated by calcineurin (e.g., Had1 and Crz1) or by another important TF, Gat201 (e.g., Opt1, Liv3, and Zfc3) ( 60 , 74 , 75 ). Finally, the expression of PDR802 itself is regulated by the TFs Gat201 and Hob1 ( 67 , 75 ).…”

Short homology-directed repair using optimized Cas9 in the pathogen Cryptococcus neoformans enables rapid gene deletion and tagging

“…We found that a Cryptococcus-optimized mNeonGreen performed particularly well. We attached to this sequence a calmodulin-binding protein tag followed by 2X FLAG purification tag we have used in prior work (DUMESIC et al 2013;DUMESIC et al 2015a;DUMESIC et al 2015b;BURKE et al 2018;CATANIA et al 2020;SUMMERS et al 2020) to produce a combined localization and purification (LAP) tag. We also appended a cleavagepolyadenylation/transcription termination sequence from the CNAG_07988 gene and a NAT1 selectable marker (MCDADE AND COX 2001).…”

“…To test our LAP tag in other cellular contexts, we tagged several additional genes selected based on information on their orthologs in Saccharomyces cerevisiae or studies of localization in C. neoformans. These genes encode the mitochondrial co-chaperone Mrj1 (HORIANOPOULOS et al 2020), the plasma membrane ATPase Pma1 (FARNOUD et al 2014), the zinc finger transcription factor Cqs2 (TIAN et al 2018;SUMMERS et al 2020) and an ortholog of the S. cerevisiae endoplasmic reticulum chaperone Erj5 (CARLA FAMA et al 2007). All of these yielded the expected localization patterns (Fig.…”