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

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 <i>Cryptococcus</i>, mutations in the <i>UXS1</i> 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 <i>Cryptococcus gattii</i>, including growth rate, ability to grow under various stress conditions and resistance to fluorinated pyrimidine analogs. RNA-seq analyses of the <i>uxs1Δ</i> mutant identify three acid protease genes, notably <i>PEP401</i>, that are differentially expressed. The absence of <i>PEP401</i> in the <i>uxs1Δ</i> background significantly reduces UDP-GlcUA levels and reverts all the phenotypes of the <i>uxs1Δ</i> mutant to the wild-type characteristics. High levels of UDP-GlcUA not only regulate expression of <i>PEP401</i> at RNA and protein levels but also enhance the proteolytic activity of total protein extracts in a <i>PEP401</i>-dependent manner, establishing a functional link between nucleotide sugar metabolism and proteolytic regulation. Moreover, the UDP-GlcUA transporter gene, <i>UUT1</i>, can further modulate the levels of UDP-GlcUA in the <i>uxs1Δ pep401Δ</i> double mutant and manifests drug resistance phenotypes observed in the <i>uxs1Δ</i> 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.