The Candida albicans KRE9 gene is required for cell wall beta-1, 6-glucan synthesis and is essential for growth on glucose

Abstract

We have isolated CaKRE9, a gene from Candida albicans, that is a functional homologue of the Saccharomyces cerevisiae KRE9 gene involved in beta-1,6-glucan synthesis. Disruption of the CaKRE9 gene in C. albicans shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of CaKRE9 grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the CaKRE9 gene product is a potentially useful candidate as a target for fungal-specific drugs.

Figure 1

Comparison of the sequence of Kre9p from C. albicans and Kre9p and Knh1p from S. cerevisiae. The amino acid residues are shown in single-letter amino acid code. Sequences were aligned with gaps to maximize homology. Dots represent a perfect match between all three sequences whereas a vertical slash indicates a conservative substitution.

Figure 2

Complementation of the growth (A) and killer resistance (B) phenotypes of the S. cerevisiae kre9Δ null mutant by the C. albicans KRE9 gene. Wild-type cells, a kre9Δ mutant, or the same mutant harboring KRE9 or CaKRE9 on multicopy vector YEp352 were scored for growth and for killer resistance in a seeded plate assay.

Figure 3

Disruption of Candida albicans KRE9 gene. (A) Schematic representation of the disruption strategy (see Materials and Methods). (B) Results of the Southern blot verifications of the correct integration of the hisG-URA3-hisG disruption module into the CaKRE9 gene and proper CaURA3 excision after fluoroorotic acid treatment. Extracted genomic DNAs are from CAI4 wild-type cells (lane 1), CaKRE9/cakre9Δ∷hisG-URA3-hisG heterozygous mutant (lane 2), CaKRE9/cakre9Δ∷hisG heterozygous mutant obtained after fluoroorotic acid treatment (lane 3), and a cakre9Δ∷hisG/cakre9Δ:hisG-URA3-hisG homozygous null mutant that was grown on galactose (lane 4).

Figure 4

Detection of cell wall β-1,6-glucan levels in wild-type and cakre9Δ mutants grown by using galactose as the sole carbon source. A specific anti-β-1,6-glucan antiserum was used to detect whole cell alkali-soluble β-1,6-glucan of different C. albicans strains by Western blotting followed by an enhanced chemiluminescence procedure (see Materials and Methods).

Figure 5

Induction of hyphal formation of different C. albicans strains. CAI4 wild-type, heterozygous mutant, and homozygous mutant cells were induced to form hyphae in presence of galactose and 20% fetal bovine serum at 37°C for up to 18 h. Cells were visualized by Nomarski optics.