Expression of 1,3-β-glucan synthase subunits in Candida glabrata is regulated by the cell cycle and growth conditions and at both transcriptional and post-transcriptional levels

Fungal cell wall-synthesizing enzyme 1,3-β-glucan synthase (GS) is the target of the echinocandins, a frontline antifungal drug class. However, increasing echinocandin resistance due to mutations in GS has been observed in certain fungal pathogens, notably Candida glabrata, where GS is encoded by two homologous genes, FKS1 and FKS2. Despite the importance of GS in the fungal life cycle and as a drug target, the regulation of its expression in culture and in the host is still poorly understood. In this study, we used a fluorescent transcriptional reporter, quantitative reverse-transcriptase PCR, protein analysis, and mining of RNA-seq data sets to examine the regulation of C. glabrata GS expression. We determined that FKS1 and FKS2 promoter activities peak during S-phase and that during exponential growth in culture, FKS1 is expressed at higher levels than FKS2. Interestingly, although FKS2 mRNA expression appeared to be strongly induced in an fks1∆ mutant, this calcineurin-mediated induction was not accompanied by increased FKS2 promoter activity, suggesting post-transcriptional regulation. Examination of FKS2 transcript across the ORF as well as Fks2 protein levels was consistent with post-transcriptional regulation. Finally, RNA-seq data mining revealed that, in contrast to vegetative growth, during the stationary phase and under host conditions, FKS2 is expressed at equivalent or higher levels than FKS1. Together, these experiments revealed that in C. glabrata, the expression of both GS subunits is regulated transcriptionally by the cell cycle, that FKS2 expression is regulated post-transcriptionally by calcineurin and fks1∆, and that in the host, FKS2 may be the more abundant subunit.