Overexpression of Sbe2p, a Golgi protein, results in resistance to caspofungin in Saccharomyces cerevisiae

Abstract

Caspofungin inhibits the synthesis of 1, 3-beta-D-glucan, an essential cell wall target in fungi. Genetic studies in the model yeast Saccharomyces cerevisiae have shown that mutations in FKS1 and FKS2 genes result in caspofungin resistance. However, direct demonstration of the role of gene overexpression in caspofungin resistance has been lacking. We transformed wild-type S. cerevisiae with an S. cerevisiae URA3-based GAL1 cDNA library and selected transformants in glucose synthetic complete plates lacking uracil (glucose SC minus uracil plates). We then moved the transformants to galactose SC minus uracil plates containing caspofungin (1 microg/ml) and looked for caspofungin-resistant colonies. We retested the candidates (true positives were sensitive on glucose caspofungin and resistant on galactose caspofungin media, respectively). We identified 16 caspofungin-resistant candidates. Restriction analysis and hybridization confirmed that 15 of the 16 clones were identical. We sequenced one of the cDNA clones and found that it contained the cDNA for SBE2. SBE2 has been described in S. cerevisiae to encode a Golgi protein involved in the transport of cell wall components (B. Santos and M. Snyder, Mol. Biol. Cell, 11:435-452, 2000). The SBE2 cDNA plasmid conferred again galactose-dependent caspofungin resistance when transformed back into the wild-type S. cerevisiae. Finally, the SBE2 deletion mutant was hypersensitive to caspofungin. In conclusion, overexpression of Sbe2p under the regulated control of the GAL1 promoter results in caspofungin resistance in S. cerevisiae. This transport pathway may provide insight into the tolerance or lack of sensitivity to caspofungin of some pathogenic fungi.

FIG. 1.

GAL1-SBE2 cDNA confers galactose-dependent resistance to CAS. (A) Growth inhibition responses of the control 10560-14C strain transformed by the GAL1 URA3 centromeric plasmid pRS 316 (control-right) and the same vector containing the SBE2 cDNA (left) on glucose and galactose SC minus uracil agar plates in the presence or absence of CAS. (B) Disk diffusion assays of the 10560-14C strain transformed by the GAL1 URA3 centromeric plasmid pRS 316 (top panels) and the same vector containing the SBE2 cDNA (bottom panels) on glucose and galactose SC minus uracil agar plates. The disks contained 5 μg of CAS. (C) Growth curves of the 10560-14C strain transformed by the GAL1 URA3 centromeric plasmid pRS 316 (control) and the same vector containing the SBE2 cDNA on glucose and galactose SC minus uracil agar liquid medium in the presence or absence of CAS (1 μg/ml).

FIG. 2.

Galactose-dependent expression of SBE2 cDNA reduces cellular swelling and vacuolization in the presence of CAS. Control (left) and GAL1-SBE2 (right) S. cerevisiae strains were grown on galactose agar plates for 48 h at 30°C in the absence (top) or presence (bottom) of CAS. DIC microscopy and image analysis were performed as described in Materials and Methods.

FIG. 3.

Galactose-dependent expression of SBE2 cDNA reduces CAS-induced cell wall damage. Control (left) and GAL1-SBE2 (right) S. cerevisiae strains were grown on galactose (top panel) and glucose (lower panel) minimal medium agar plates for 48 h at 30°C in the absence (top) or presence (bottom) of CAS. Transmission electron microscopy was performed as described in Materials and Methods.

FIG. 4.

Galactose-dependent expression of SBE2 cDNA increases the baseline glucan synthase activity and decreases its degree of inhibition by CAS. Control and GAL1-SBE2 S. cerevisiae strains were grown on glucose (A) and galactose (B) SC minus uracil liquid media. Membrane fractions were harvested, different concentrations of CAS were added, and glucan synthase activity was measured as described in Materials and Methods. The mean and standard deviation (error bars) of the three experiments, after first averaging the replicates in each experiment, are plotted. The statistical significance of the measurements of the control strain versus the SBE2-overexpressing strain is shown. Comparisons were performed, at each concentration separately, using two-sample t tests.