Cwh8 Moonlights as a Farnesyl Pyrophosphate Phosphatase and is Essential for Farnesol Biosynthesis in Candida albicans

In the opportunistic pathogen Candida albicans, hyphal growth and virulence factor expression are regulated by environmental and chemical cues. Farnesol is a secreted autoregulatory molecule that represses filamentation. It is derived from farnesyl pyrophosphate (FPP), an ergosterol biosynthesis pathway intermediate. Although Dpp1, Dpp2, and Dpp3 were proposed to synthesize farnesol, a mutant lacking all three had only a modest reduction in farnesol. To identify other farnesol biosynthesis genes, we employed transcription factor mutants that underproduced or overproduced farnesol in a screen and analyzed their transcriptomes. CWH8 was the only transcript correlated with farnesol production. Cwh8 is a lipid phosphatase known to recycle dolichyl pyrophosphate during glycosylation of cell wall proteins. The cwh8?? mutant had a >99% reduction in farnesol compared to the parent strain and was hyperfilamentous in embedded conditions. It had ~900-fold lower levels of farnesyl phosphate, the product of FPP dephosphorylation, suggesting Cwh8 acts directly on FPP. Complementation of the C. albicans cwh8?? mutant with CWH8 from C. albicans restored cell wall integrity and farnesol production. However, complementation with CWH8 from Clavispora lusitaniae, a species lacking farnesol signaling, restored cell wall integrity but failed to rescue farnesol production. This indicates that while Cwh8's role in maintaining cell wall integrity is conserved, differences in substrate specificity or interactions with species-specific cofactors underlie its involvement in farnesol biosynthesis in C. albicans. Finally, fluconazole was fungicidal for cwh8?? rather than fungistatic. These studies demonstrate how an extracellular signaling system can arise and be co-opted to promote fungal fitness in complex environments. Overall design: To identify genes involved in the synthesis of farnesol we performed an RNA-seq experiment on two farnesol underproducing transcription factor deletion mutants, msn4 and cas5 that were identified from previous work by our group (Gutzmann et al. 2023 G3). We additionally performed two additional RNA seq experiments, one comparing MAY1375 and cwh8?? cells, and another comparing MAY1375, pTDH3-CWH8 cells, and MAY1375 + 50µM FOH treated cells.