Filamentation and Biofilm Formation are Regulated by the Phase-Separation Capacity of Network Transcription Factors in Candida albicans

The ability of the fungus Candida albicans to filament and form biofilms contributes to its burden as a leading cause of hospital-acquired infections. Biofilm development involves an interconnected transcriptional regulatory network (TRN) consisting of nine transcription factors (TFs) that bind both to their own regulatory regions and to those of the other network TFs. Here, we show that seven of the nine TFs in the C. albicans biofilm network contain prion-like domains (PrLDs) that have been linked to the ability to form phase-separated condensates. Construction of PrLD mutants in four biofilm TFs reveals that these domains are essential for filamentation and biofilm formation in C. albicans. Moreover, biofilm PrLDs promote the formation of phase-separated condensates in the nuclei of live cells, and PrLD mutations that abolish phase separation (such as the removal of aromatic residues) also prevent biofilm formation. Biofilm TF condensates can selectively recruit other TFs through PrLD-PrLD interactions and can co-recruit RNA polymerase II, implicating condensate formation in the assembly of active transcriptional complexes. Finally, we show that PrLD mutations that block the phase separation of biofilm TFs also prevent filamentation in an in vivo model of gastrointestinal colonization. Together, these studies associate transcriptional condensates with the regulation of filamentation and biofilm formation in C. albicans, and highlight how targeting of PrLD-PrLD interactions could prevent pathogenesis by this species. Overall design: C. albicans biofilm TF PrLDs were removed or mutated to determine their contribution to biofilm formation, filamentation, and phase separation. Efg1 and Brg1 WT and YF-to-S mutants were grown under filament-inducing conditions, Spider medium at 37°C for 6 h, and measured for yeast vs. hyphal cells and harvested for RNA. RNA sequencing was performed on all strains (in triplicate) to measure gene expression.