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.