Two closely related β-1,2-xylosyltransferases differentially impact fungal glycan synthesis

Cryptococcus neoformans is an opportunistic fungal pathogen that causes pulmonary infection in immunocompromised patients, which in severe cases leads to fatal meningoencephalitis. Cryptococcus exhibits unique glycobiology that plays important roles in pathogenesis. Unlike model yeast and other common fungal pathogens, Cryptococcus incorporates xylose, a five-carbon monosaccharide, into its glycans. One trimer motif, which consists of xylose in β-1,2 linkage to the reducing mannose of an α-1,3-mannose dimer, occurs in key cryptococcal glycoconjugates that include protein N- and O-linked glycans, glycosylinositol phosphorylceramides (GIPCs), and the capsule polysaccharides glucuronoxylomannan (GXM) and glucuronoxylomannogalactan (GXMGal). We previously identified cryptococcal β-1,2-xylosyltransferase 1 (Cxt1), which catalyzes formation of this motif in GIPCs, GXM, and GXMGal. Here, we report the discovery of a second enzyme, cryptococcal β-1,2-xylosyltransferase 2 (Cxt2). Through characterization of cells that lack one or both corresponding genes (CXT1 and CXT2), we have dissected the biological roles of these enzymes, which are overlapping but not identical. Notably, Cxt1 and Cxt2 co-localize in the Golgi, influence capsule in a strain-dependent manner, and together are responsible for all xylose addition to O-glycans. Overall, our work highlights unique roles of these two enzymes and fills a gap in understanding of cryptococcal glycan synthesis.