Identification of an N-acetylglucosamine transporter that mediates hyphal induction in Candida 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.

Figure 1.

Ngt1-GFP induction by GlcNAc. C. albicans strain YJA1 carrying an NGT1-GFP fusion gene was grown in the indicated medium for 2 h at 37°C, and then fluorescence and light microscope images were recorded. Cells were incubated in minimal medium containing glucose, GlcNAc, or glucose plus 10% bovine serum as indicated. Fluorescent microscope images of Ngt1-GFP are shown in the top panels and light microscope (DIC) images are shown below. Bar, 10 μm.

Figure 2.

Ngt1-GFP is specifically induced by GlcNAc and is repressed by glucose. (A) Relative fluorescence of cells incubated in medium containing the indicated sugar for 4 h. For each sugar, the first bar represents the NGT1-GFP strain YJA1, and the second bar is the untagged strain SC5314. Fluorescence was very low under all conditions for the untagged strain. (B) Relative fluorescence of cells induced with the indicated sugar or combination of GlcNAc and another sugar. Note that glucose repressed induction of NGT1-GFP. Relative fluorescence was determined from analysis of digital microscope images (see Materials and Methods).

Figure 3.

Ngt1-GFP is induced after macrophage phagocytosis. The murine macrophage cell line J774 was infected with C. albicans NGT1-GFP strain YJA1 or wild-type strain SC5314 at an MOI of 1 for 40 min. The cells were incubated for 2 h and then fixed with paraformaldehyde. Fluorescent microscope images of Ngt1-GFP are shown on top and light microscope (DIC) images are shown below. Bar, 10 μm.

Figure 4.

NGT1 is required for growth on GlcNAc medium. Wild-type strain SC5314, ngt1Δ homozygous deletion strain YJA3, and strain YJA4 in which ngt1Δ was complemented by reintegration of NGT1, were adjusted to 5 × 10⁶ cells/ml, and then 10-fold serial dilutions of cells were spotted onto solid medium plates containing either glucose or GlcNAc. The plates were incubated for 2 d at 30°C and then photographed.

Figure 5.

High levels of GlcNAc can bypass the role of Ngt1 in stimulating hyphal growth. Wild-type strain DIC185, ngt1Δ homozygous deletion strain YJA3, and strain YJA4 in which ngt1Δ was complemented by reintegration of NGT1 were grown to log phase, resuspended in medium containing the indicated concentration of GlcNAc, and then incubated at 37°C for 2 h. Hyphal cells clumped, presumably due to induction of the cell surface adhesin proteins. Bar, 10 μm.

Figure 6.

NGT1 is needed for efficient GlcNAc uptake by C. albicans. (A) Wild-type strain SC5314, (B) ngt1Δ homozygous deletion strain YJA3, and (C) strain YJA4 in which ngt1Δ complemented by reintegration of NGT1 were incubated for 4 h at 30°C in minimal medium containing GlcNAc (▲) to induce NGT1, and were also grown in glucose medium (■) as a control. Cells were harvested and washed, and then 10⁷ cells were then incubated in 200 μM [³H]GlcNAc for the indicated time. Cells were collected on filters, and the [³H]GlcNAc taken up was determined by scintillation counting. Error bars, SE.

Figure 7.

NGT1 expression in S. cerevisiae is sufficient to promote GlcNAc uptake. S. cerevisiae strain W303 carrying a plasmid designed to (A) express NGT1 under control of the galactose-inducible promoter GAL1 or (B) the empty vector plasmid (pRS426GAL1). Cells were grown in galactose medium (▲) overnight to induce NGT1, or in glucose medium (■) to repress NGT1. Cells (n = 10⁷) were incubated with 200 μM [³H]GlcNAc for the indicated time and then the [³H]GlcNAc taken up was determined by scintillation counting. (C) Fluorescent microscope image and corresponding light microscope image of S. cerevisiae cells induced with galactose to express NGT1-GFP. Variable levels of Ngt1-GFP observed in different cells are expected due the cells containing different levels of the multicopy plasmid vector pRS426GAL1 used to express NGT1-GFP. Error bars, SE.

Figure 8.

Ngt1 is specific for GlcNAc uptake. The specificity of the transport activity promoted by Ngt1 was examined by testing the ability of other sugars to compete with the uptake of [³H]GlcNAc. Assays were carried out with S. cerevisiae strain W303 that expressed NGT1 under control of the galactose-inducible promoter GAL1 as described in the legend to Figure 7. Reactions contained 200 μM [³H]GlcNAc plus either 2 mM or 20 mM of the indicated nonradioactive sugar and were carried out for 2 min. The mock sample received only water. The results demonstrate that only nonradioactive GlcNAc, and not other related sugars, competed for the uptake of [³H]GlcNAc. Error bars, SE.

Figure 9.

Evolutionary conservation of NGT1. BLAST searches (Altschul et al., 1990) were used to identify NGT1 homologues in the genome sequences of other organisms. White letters in black boxes indicate the presence of NGT1 and black letters indicate the absence. *S. kluyveri is marked by an asterisk because it lacks NGT1 and DAC1, but contains NAG1. **S. pombe is marked because it contains an NGT1 homolog but lacks the other catabolic genes (e.g., DAC1 and NAG1). ***P. carinii contains an NGT1 homolog, but the genome sequence is not complete so it is not clear if homologues of NAG1 and DAC1 will be present. The evolutionary tree was constructed based on a recent phylogenetic analysis of fungi (James et al., 2006) and also on an analysis of Ascomycota (Dujon, 2006). The branches in the figure were sized to clearly display the relationship of the different lineages and are not sized to represent predicted evolutionary distance.