Role of three chitin synthase genes in the growth of Candida albicans

Abstract

The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2⌬ and homozygous chs3⌬ null mutants.Like Saccharomyces cerevisiae, Candida albicans harbors three chitin synthase genes, designated CHS1, CHS2, and CHS3 (2, 6, 13). In S. cerevisiae, it was demonstrated by gene disruption experiments that chitin synthase 1 (Chs1p) is involved in the repair of damaged chitin, Chs2p is required for primary septum formation, and Chs3p is responsible for all other chitin syntheses (5,12,14). More recently, Kollar et al. reported that CHS3 also contributes to the formation of linkage between chitin and ␤-1,3-glucan in S. cerevisiae (10). In order to gain more insights into the physiological roles of the chitin synthases of C. albicans, we have disrupted both CHS2 and CHS3 in C. albicans by means of the URA blaster protocol (1).The homozygous chs2⌬ null mutant and the homozygous chs3⌬ null mutant strains of C. albicans were obtained by transforming CAI-4 cells (ura3⌬::imm34/ura3⌬::imm34) with DNA fragments containing either CHS2 in which the hisG-URA3-hisG cassette was inserted at the unique XhoI site or CHS3 in which the 0.8-kb NcoI-ClaI region was replaced by the hisG-URA3-hisG cassette by the lithium acetate method (9). These DNA fragments were successfully integrated into one of the diploid CHS2 or CHS3 alleles, respectively, and the URA3 gene was efficiently eliminated by 5-fluoroorotic acid (5-FOA) selection (11) (Fig. 1). Then these DNA fragments were again transfected into cells in which one of the diploid CHS2 or CHS3 alleles was already flanked by the hisG sequence. Although the second allele of the CHS2 locus was efficiently targeted by the same DNA fragment used to disrupt the first allele, the remaining CHS3 allele was not easily disrupted by transfection of the same DNA fragment. Therefore, we constructed another plasmid in which the hisG-URA3-hisG cassette was inserted at the NcoI site of CHS3. We assumed that use of this DNA for the second round of transfection would increase the efficiency of homologous recombination between the transfected DNA and the remaining intact CHS3 allele because the 0.8-kb NcoI-ClaI region of CHS3 was missing in the already targeted CHS3 locus. As expected, in 3 of 24 uracil auxotrophs, both of the CHS3 alleles were found to be flanked by the hisG sequence after 5-FOA selection, resulting in the homozygous chs3⌬ null mutation (Fig. 1).Cells lacking functional CHS3 grew in a rich medium such as YPD (1% peptone, 2% yeast extract, and 2% dextrose), but their growth was somewhat slower than that of cells missing CHS2 or the parental strain CAI-4 (the doubling times for CAI-4, the homozygous chs2⌬ null mutant, and the homozygous chs3⌬ null mutant were about 70, 72, and 90 min, respectively)...

Inhibition of Chitin Synthases and Antifungal Activities by 2'-Benzoyloxycinnamaldehyde from Pleuropterus ciliinervis and Its Derivatives

“…The structure of isolated compound was determined by EI-MS and various NMR spectroscopic analyses including 1 H, 13 Although several chitin synthase inhibitors have been isolated from microbes and higher plants, [28][29][30][31][32] 2Ј-BCA has not yet been reported as chitin synthase inhibitor. The inhibitory activities of this compound on chitin synthase isozymes were investigated by the filter binding assay using UDP-14 C-Glc-NAC as substrate.…”

The Candida albicans CHS4 gene complements a Saccharomyces cerevisiae skt5/chs4 mutation and is involved in chitin biosynthesis

“…Nevertheless, chs3-deficient C. albicans still produced hyphae (Bulawa et al, 1995;Mio et al, 1996), and therefore expression of the CHSS gene did not determine the morphology. To examine the morphological transition in the chs4 mutant, we tested the C. albicans chs4 mutant in the RPMI 1640 medium that was used for the morphological analysis of S. cereuisiae chs mutants.…”

Inhibitory effect of nikkomycin Z on chitin synthases in Candida albicans

“…Among them, CaChs3, involved in cell wall synthesis, is the only one with clear cellular function (Mio et al, 1996). Since the null Cachs1D/ Cachs1D mutant is yet to be obtained, in contrast to availability of Scchs2D/Scchs2D mutant in S. cerevisiae, it has been suggested that CaChs1 is an essential gene for cell growth.…”