IDENTIFICATION OF THE FKS1 GENE OF CANDIDA-ALBICANS AS THE ESSENTIAL TARGET OF 1,3-BETA-D-GLUCAN SYNTHASE INHIBITORS

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhib itors. A 2.1-kb portion of this gene, designated CaFKS1, has significa nt homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate t he role of CaFks1p in susceptibility to echinocandins, we disrupted Ca FKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been sel ected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ec h(r)) or fully sensitive (Ech(s)) to inhibition by L-733,560 in both l iquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, u sing restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transf ormant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transform ant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygou s for a dominant or semidominant pneumocandin resistance mutation at C aFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allel e, and (iii) CaFks1p is a target of the echinocandins. For transforman ts of strain NR3, all the clones we analyzed were uniformly Ech(r), an d only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain a n Ech(r) mutation. Transformants derived from the mutants were analyze d for susceptibility to pneumocandin treatment in a mouse model of dis seminated candidiasis. Strains heterozygous for the resistant allele ( i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to t reatment, while strains without a functional Ech(s) allele (i.e., stra in NR3 and derivatives of strain CAI4R1 with the disruption plasmid in tegrated in the Ech(s) allele) displayed strong in vivo echinocandin r esistance. Finally, we were unable to inactivate both alleles at CaFKS 1 by two-step integrative disruption, suggesting that CaFks1p is likel y to be an essential protein in C. albicans.