Expression profiling of Histoplasma capsulatum G217B yeast to filament transition in GlcNAc or glucose for wild type, msb2, and complemented strains

Dimorphic fungi are temperature-sensitive organisms that couple their cell shape with their environment. One of these fungi, Histoplasma capsulatum, exists as both a soil-dwelling hypha and a host-associated yeast. Here we examine the role of the previously uncharacterized gene MSB2 in filamentous growth. We performed a genetic screen to identify insertion mutants that are unable to transition from the yeast form to the hyphal form. One yeast-locked mutant has an insertion upstream of the MSB2 gene. This mutant strain (SG1) fails to express MSB2, whose ortholog in the model yeast Saccharomyces cerevisiae is a known signaling component in the high osmolarity glycerol (HOG) pathway and filamentous growth (FG) pathway. Here, we profiled gene expression by RNA-seq to characterize transcriptional differences between wild type and msb2 mutant strains during the yeast to hyphal transition. Overall design: Samples were were prepared in two distinct experiments and sequenced in three distinct sequencing runs. In the first experiment, two biological replicates each of Histoplasma capsulatum G217B ura5 (WU15) or an msb2 mutant derived from WU15 (SG1) were grown in liquid culture with glucose or GlcNAc as the carbon source at 37C. After taking t=0 samples, the cultures were moved to room temperature (RT) and additional samples were taken at 2, 6, 8, or 10 days. One sample was not usable for sequencing, giving a total of 2*2*2*5-1 = 39 samples. The GlcNAc samples were sequenced in part of a lane of one HiSeq 4000 run (batch 1) and the glucose samples were sequenced in part of a lane of a second run (batch 2). Because these samples were grown at the same time, they were treated as a single batch in our limma analysis. In the second experiment, two biological replicates each of WU15, SG1, or SG1 episomally complemented with MSB2::PaURA5 (SG1cMSB2) were grown in liquid culture with glucose or GlcNAc as the carbon source at 37C. After taking t=0 samples, the cultures were moved to RT and additional samples were taken at 2, 6, or 8 days, for a total of 2*3*2*4 = 48 samples. These samples were sequenced in a full lane of a HiSeq 4000 run (batch 3). In this second experiment, WU15 and SG1 were episomally complemented with PaURA5 to control for the selectable PaURA5 marker in the SG1cMSB2 strain.