Abstract Candida glabrata is a clinically significant cause of candidemia, yet how it supplies uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) for cell-wall chitin synthesis remains unclear. Here, we identify an N-acetylglucosamine (GlcNAc) kinase encoded by CAGL0M00682 g and designated CgNgk1. In vitro, purified CgNgk1 preferentially phosphorylated GlcNAc with markedly higher catalytic efficiency than other sugars and accepted multiple nucleoside triphosphates, including ATP, as phosphoryl donors. Site-directed mutagenesis indicated that Asp197 and Lys155 are required for catalysis and ATP binding, respectively. In vivo, CgNgk1 overexpression in C. glabrata increased intracellular UDP-GlcNAc in a GlcNAc dose-dependent manner, whereas the kinase-dead D197N variant had no effect. CgNgk1-mediated GlcNAc phosphorylation elevated cell-wall chitin and heightened sensitivity to sodium dodecyl sulfate, heat, and low pH, but did not affect β-1,3-glucan–related phenotypes such as zymolyase or caspofungin sensitivity. Under GlcNAc supplementation, CgNgk1-overexpression specifically decreased cellular susceptibility to flucytosine, while responses to voriconazole and amphotericin B were unchanged. Overexpression of the putative hexosamine-pathway regulator CgIsr1 lowered intracellular UDP-GlcNAc, consistent with an intact endogenous pathway. These findings demonstrate that imported GlcNAc is routed via CgNgk1 to UDP-GlcNAc to promote chitin biosynthesis, and that quantitative control of this route shapes cell-wall integrity and modulates responses to host-relevant stresses and antifungal agents.
Yamada et al. (Fri,) studied this question.