Cwh8 moonlights as a farnesyl pyrophosphate phosphatase and is essential for farnesol biosynthesis in .

In the opportunistic pathogen , 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. 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 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 mutant with from restored cell wall integrity and farnesol production. However, complementation with from , 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 . Finally, fluconazole was fungicidal for rather than fungistatic. These studies demonstrate how an extracellular signaling system can arise and be co-opted to promote fungal fitness in complex environments.IMPORTANCEFarnesol secretion distinguishes the human opportunistic pathogen from non-secreting, non-pathogenic yeasts. Despite 20 years of research, surprisingly little is known about how farnesol is synthesized and regulated. Using transcriptomic profiles from mutants with altered farnesol production, we identified as a critical enzyme in this process. null mutants produced no farnesol. Our novel assay measuring cellular farnesyl pyrophosphate (FPP) and farnesyl phosphate (FP) showed that Cwh8 converts FPP to FP in the first step of farnesol biosynthesis, in addition to its established role in recycling dolichyl pyrophosphate. Farnesol-secreting fungi had huge metabolic pools of FP, while nonsecretors had small pools. Furthermore, expressing from , a non-farnesol-secreting species, failed to restore farnesol production in . This suggests that changes in Cwh8 enzyme specificity drove the evolution of farnesol as a signaling molecule and virulence factor in .