Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors

Abstract

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 inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using 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) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and 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 an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

Antifungal drug resistance of oral fungi

“…51 The incidence of 5-FC resistance in fungi means it is primarily used in combination with other antifungals such as AMB. 52 There is a low incidence of echinocandin resistance in clinical isolates of Candida species that are normally sensitive to echinocandins, despite the ready in vitro selection of echinocandin-resistant variants of C. albicans 53,54 or S. cerevisiae. 55 Echinocandin-resistant Candida isolates usually have single amino acid point mutations in the β(1,3)-glucan synthase subunit (Gsc1p) that is orthologous to S. cerevisiae Fks1p.…”

Diagnosis of Antifungal Drug Resistance Mechanisms in Fungal Pathogens: Transcriptional Gene Regulation

“…Target alterations have been observed, conferring echinocandin resistance at the level of the enzyme β-1,3 glucan synthase (FKS1), and similar mutations were obtained by in vitro selection after exposure to the drug [41,42]. These mutations are located in two hot-spot regions (HS1, HS2), though HS1 (located between residues 641 and 649 of the C. albicans Fks1) is the region with most substitutions [9].…”

Pharmacology, in vitro activity, and in vivo efficacy of new antifungal agents

“…This inhibits the production of (1→3)-β-D-glucan, a major and essential component of the fungal cell wall that contributes to its shape and integrity [43]. Depletion of these polysaccharides within the cell wall results in osmotic instability and cell wall lysis in many pathogenic fungi [43][44][45]. This is an attractive target for antifungal activity because human cells lack a homologous enzyme; thus, signifi cant collateral toxicities and drug interactions associated with the azoles and amphotericin B are avoided.…”

“…1) that binds to and noncompetitively inhibits the (1→3)-β-D-glucan synthase enzyme located within the cell membrane [22,43]. This inhibits the production of (1→3)-β-D-glucan, a major and essential component of the fungal cell wall that contributes to its shape and integrity [43]. Depletion of these polysaccharides within the cell wall results in osmotic instability and cell wall lysis in many pathogenic fungi [43][44][45].…”

“…Like the other members of the echinocandin class, aminocandin is a large cyclic semisynthetic lipopeptide (Fig. 1) that binds to and noncompetitively inhibits the (1→3)-β-D-glucan synthase enzyme located within the cell membrane [22,43]. This inhibits the production of (1→3)-β-D-glucan, a major and essential component of the fungal cell wall that contributes to its shape and integrity [43].…”