Elevated UDP-glucuronic acid levels mend drug resistance and stress responses via a protease and a transporter in Cryptococcus gattii

UDP-glucuronic acid (UDP-GlcUA) is a nucleotide sugar essential for various biological processes in many organisms, and its excess within the cell can disrupt cellular functions. In Cryptococcus , mutations in the UXS1 gene which encodes an enzyme responsible for converting UDP-GlcUA into UDP-xylose, result in excessive accumulation of UDP-GlcUA and confer resistance to the antifungal drug 5-fluorocytosine. Here, we demonstrate that elevation of UDP-GlcUA affects several cellular processes in Cryptococcus gattii , including growth rate, ability to grow under various stress conditions and resistance to fluorinated pyrimidine analogs. RNA-seq analyses of the uxs1Δ mutant identify three acid protease genes, notably PEP401 , that are differentially expressed. The absence of PEP401 in the uxs1Δ background significantly reduces UDP-GlcUA levels and reverts all the phenotypes of the uxs1Δ mutant to the wild-type characteristics. High levels of UDP-GlcUA not only regulate expression of PEP401 at RNA and protein levels but also enhance the proteolytic activity of total protein extracts in a PEP401 -dependent manner, establishing a functional link between nucleotide sugar metabolism and proteolytic regulation. Moreover, the UDP-GlcUA transporter gene, UUT1 , can further modulate the levels of UDP-GlcUA in the uxs1Δ pep401Δ double mutant and manifests drug resistance phenotypes observed in the uxs1Δ mutant. Collectively, these findings reveal a previously unrecognized regulatory network that links UDP-GlcUA metabolism to protease-mediated cellular processes and the transport of UDP-GlcUA. This interaction provides a foundation for targeting nucleotide sugar metabolism and protease regulation in the development of enhanced therapeutic strategies against cryptococcosis.