Figure 1.
Identification of Snf3/Rgt2 homologs in Cryptococcus.
A. Phylogenetic tree of proteins from C. albicans (Hgt4 and Hgt12) and C. neoformans (Hxs1 and Hxs2) that share high homology with glucose sensors Snf3 and Rgt2 in S. cerevisiae. The full-length protein sequences were used for alignment using ClustalX. The hexose transporter Hxt1 in S. cerevisiae was used as an out-group. B. Predicted topology of the deduced Hxs1 amino acid sequence based on the TMpred program. Hydropathy values are on the y-axis, and the residue numbers are on the x-axis. The predicted transmembrane domains (TM1 to 12) are numbered. C. Schematic models of the primary protein structures of Snf3, Rgt2, Hxs1 and Hxs2. The black boxes represent transmembrane regions. The total number of amino acids for each protein and its C-terminal tail are indicated.
Figure 2.
The expression of HXS1 is repressed by glucose.
The qRT-PCR method was used to detect the change in transcription levels of HXS1 and HXS2 under growth conditions with different glucose levels. H99 cells were grown on YPG (no glucose) liquid medium, then switched to medium with 0.01%, 0.1%, 1% or 2% glucose (A), or grown on YPD and switched to medium with lower glucose levels (1%, 0.1%, 0.01% or YPG) (B). Cells were collected after 2 hr incubation and RNA prepared for qRT-PCR. Values are expressed as relative expression (log2) of the HXS1 or HXS2 gene, normalized to the GAPDH gene endogenous reference. The changes in gene transcription levels were related to 0-hr time point (H99 overnight liquid cultures on either YPG (A) or YPD (B)). The error bars showed standard deviations of three repeats.
Figure 3.
Hxs1 is not required for the expression of other hexose transporter homologs.
The qRT-PCR method was used to measure the expression of seven hexose transporter homologs HXT1-7 (A–G) and SUC2 (H) in C. neoformans under YPG, YP with 0.1% glucose, or YPD (2% glucose) growth conditions. Values are expressed as relative expression of these genes, normalized to the GAPDH gene endogenous reference, and relative to HXS1 expression in H99 on YPG medium. Error bar indicates the standard deviation of three repeats.
Figure 4.
Hxs1 is required for Cryptococcus glucose uptake and cell growth on low glucose media.
A. Glucose uptake assay was performed for Cryptococcus wild type H99, the hxs1Δ mutant or its complemented strain as described in Materials and Methods. Error bar indicates the standard deviation of three repeats. B–H. Cryptococcus cell growth was assayed in 96-well plates. 1×105 cells of each strain were inoculated into the wells containing 100 µl YP supplemented with either 2% mannitol (B), 2% sucrose (C), 2% galactose (D), 2% glucose (E), 1% glucose (F), 0.1% glucose (G), or 0.01% glucose (H). The plates were kept in a PerkinElmer precisely Envision 2014 Multilabel Reader and incubated at 30°C with shaking (350RPM) and OD600 were measured in real time every half hour. Each experiment was performed in triplicates. Error bars indicate standard deviations.
Figure 5.
Hxs1 is required for stress response.
Cultures of wild type H99, the hxs1Δ mutant and its complemented strain were inoculated on YPD with 0.05% SDS, 0.5% Congo Red, 1 M Sorbitol, 5 mM H2O2, 1 M KCl, or 1.5 M NaCl, respectively. Plates were incubated at 30°C (upper) or 37°C (lower) for 3 days and photographed.
Figure 6.
Hxs1 is required for fungal virulence.
A. Cultures of H99, hxs1Δ and its complement strain were inoculated on DME medium for capsule induction, or on L-DOPA medium for melanin induction. Plates were incubated at 30°C or 37°C for 48 hr and photographed. B. To determine the fungal virulence, female A/Jcr mice were intranasally infected with 105 cells of H99, hxs1Δ and its complement strain. Animals were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical endpoint that predicts imminent mortality.
Figure 7.
Heterologous expression of HXS1 in a Saccharomyces strain lacking hexose transporters showed high glucose uptake activity.
A. Localization of HXS1, HXS2 in S. cerevisiae was determined by overexpressing a GFP:HXS1 or GFP:HXS2 fusion protein in EBY.VW1000, a mutant lacking all hexose transporters. EBY.VW1000 expressing an empty vector was used as a control. B and C. Glucose uptake activity (B) or glucose binding activity (C) of a Saccharomyces strain expressing HXS1. Cultures of the background strain CEN.PK2.1C and EBY.VW1000 expressing the pTH74 empty vector, the HXS1, or the HXS2 genes were inoculated on SD media lacking Uracil but containing 2% maltose as carbon source. Yeast cells were mixed with 3H-labeled glucose and incubated at 30°C(B) or 0°C (C) for 1, 5, and 10 mins. This assay was repeated twice with similar patterns. D. Glucose uptake assay was performed for S. cerevisiae strains CEN.PK2.1C and EBY.VW1000 expressing the empty vector or HXS1 in the presence of 0.1% or 2% cold glucose. The error bar indicates the standard deviation of three repeats.
Figure 8.
Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant background failed to complement glucose sensor function.
A. Localization of HXS1, HXS2 in S. cerevisiae was determined by overexpressing a GFP:HXS1, GFP:HXS2, or GFP:RGT2 fusion protein in a Saccharomyces strain lacking both Rgt2 and Snf3 glucose sensors. B. The growth of S. cerevisiae wild type BY4741, the snf3Δ rgt2Δ double mutant expressing empty vector pTH74, HXS1, HXS2, and RGT2 were inoculated on YPG or YPD with 1 µg/ml antimycin A. Plates were incubated at 30°C for 3 days.
Table 1.
Strains used in this study.