Identification of anN-Acetylglucosamine Transporter That Mediates Hyphal Induction inCandida albicans

Abstract

The sugar N-acetylglucosamine (GlcNAc) plays an important role in nutrient sensing and cellular regulation in a wide range of organisms from bacteria to humans. In the fungal pathogen Candida albicans, GlcNAc induces a morphological transition from budding to hyphal growth. Proteomic comparison of plasma membrane proteins from buds and from hyphae induced by GlcNAc identified a novel hyphal protein (Ngt1) with similarity to the major facilitator superfamily of transporters. An Ngt1-GFP fusion was detected in the plasma membrane after induction with GlcNAc, but not other related sugars. Ngt1-GFP was also induced by macrophage phagocytosis, suggesting a role for the GlcNAc response in signaling entry into phagolysosomes. NGT1 is needed for efficient GlcNAc uptake and for the ability to induce hyphae at low GlcNAc concentrations. High concentrations of GlcNAc could bypass the need for NGT1 to induce hyphae, indicating that elevated intracellular levels of GlcNAc induce hyphal formation. Expression of NGT1 in Saccharomyces cerevisiae promoted GlcNAc uptake, indicating that Ngt1 acts directly as a GlcNAc transporter. Transport mediated by Ngt1 was specific, as other sugars could not compete for the uptake of GlcNAc. Thus, Ngt1 represents the first eukaryotic GlcNAc transporter to be discovered. The presence of NGT1 homologues in the genome sequences of a wide range of eukaryotes from yeast to mammals suggests that they may also function in the cellular processes regulated by GlcNAc, including those that underlie important diseases such as cancer and diabetes. INTRODUCTIONN-acetylglucosamine (GlcNAc) is an amino sugar that carries out important roles in a broad range of cells from bacteria to humans. One aspect of GlcNAc function is to mediate cellular signaling. In bacteria, GlcNAc induces components that are important for colonization of human hosts, including fimbrins that mediate adhesion to host cells (Sohanpal et al., 2004), multidrug exporter genes (Hirakawa et al., 2006) and Curli fibers that promote biofilm formation (Barnhart et al., 2006). In mammals, GlcNAc is a key sensor of nutrient status that is involved in insulin signaling, cell cycle control, and other essential processes. Nutritional effects that lead to increased GlcNAc levels result in its conversion to UDP-GlcNAc and subsequent attachment to proteins on serine or threonine residues in a dynamic manner that is analogous to modification of proteins by phosphorylation (Slawson and Hart, 2003;Zachara and Hart, 2006). In fact, the interplay between O-GlcNAc modification and phosphorylation of proteins regulates many critical transcription factors, such as c-myc and p53 (Chou and Hart, 2001;Yang et al., 2006). O-GlcNAc modification also regulates other processes including proteosome function (Zachara and Hart, 2004). In addition to these regulatory roles, GlcNAc contributes to the N-linked glycosylation, glycophosphatidylinositol (GPI) anchor addition to proteins and is polymerized into chitin, which forms part of the fungal cell wall and the exoskele...

N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

“…The signaling pathways induced by GlcNAc in C. albicans are not yet defined well. One reason for this is that the commonly studied model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the genes needed to catabolize GlcNAc (12). Also, post-translational modification of proteins by O-GlcNAc attachment is not known to occur in C. albicans as it does in humans and other metazoans.…”

“…Transport of GlcNAc into C. albicans is mediated by a recently discovered plasma membrane transporter, Ngt1, which represents the first eukaryotic GlcNAc transporter to be identified (12). Ngt1 appears to specifically transport GlcNAc, and not other related sugars.…”

Targeted metabolic labeling of yeast N-glycans with unnatural sugars

“…For example, gfa1Δ strains of S. cerevisiae are capable of internalizing and phosphorylating extracellular glucosamine, effectively bypassing the GFA1 deletion (25). Some organisms, such as C. albicans, have functional salvage pathways allowing them to internalize and phosphorylate GlcNAc (18,19,26), but S. cerevisiae lacks these activities and gna1Δ strains cannot be rescued by extracellular GlcNAc (Fig. 2).…”

“…To enable utilization of extracellular GlcNAc, we introduced plasmids carrying the recently characterized C. albicans GlcNAc transporter NGT1 into the same GNA1/gna1Δ strain (19). While NGT1 was initially cloned into the pRS41X-GPD scaffolds, no transformants could be obtained from plasmids containing GPD promoters, suggesting S. cerevisiae may be sensitive to NGT1 expression level.…”

“…1B). Specifically, we employed the recently characterized GlcNAc transporter NGT1 from Candida albicans (19) and human GlcNAc kinase NAGK. This functional salvage pathway allows bypass of the otherwise lethal GNA1 deletion so long as the cells are provided with a GlcNAcsupplement in their growth medium.…”