An association between reduced susceptibility to echinocandins and changes in the 1,3-␤-D-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

In Saccharomyces cerevisiae, mutations in FKSJ confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETGI confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-J3D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z.The FKS1 and ETGI genes were cloned by complementation of these phenotypes and were found to be identical. The immunosuppressants FK506 and cyclosporin A (CsA) also have antifungal activity. Although vegetative growth of yeast is not potently inhibited by these drugs, recovery from mating factor arrest is (8). The drugs inhibit yeast recovery and T-cell activation by similar mechanisms. Each binds to an intracellular receptor (FKBP12 for FK506 and cyclophilin for CsA), and the receptor-drug complex inhibits the Ca2+/ calmodulin-dependent protein phosphatase calcineurin (9, 10). We previously described a mutation (Jksl-l) which results in calcineurin-dependent growth and hypersensitivity to FK506 (FKs) and CsA (11). We cloned the hypersensitivity locus (FKSJ) to help identify targets of calcineurin.t To our surprise FKS1 and ETGI are identical.:MATERIALS AND METHODS Microbiological Methods and Strains. YPAD and drop-out (DO) media and procedures for mating, sporulation, tetrad analysis, transformation, gene disruption, and determination of antibiotic sensitivity have been described (6,12). Meiotic progeny of diploid YFK016 (12) were mated to produce the yeast a/a diploid YFK419 (homozygous for ade2-101 his3-A200 leu2-Al lys2-801 trpl-Al, and ura3-52). R560-1C (MATa ade2-1 canl his3-11,15 leu2-3,112 trpl-l ura3-1 etgl-l) and MS14 (MATa etgl4) are spontaneous L-733,560-resistant (EchR) mutants derived from W303-1A (6) and X2180-1A (7), respectively. EchR mutants are resistant to drug on uracil DO medium at 8 ,g/ml, whereas the wild type is sensitive at 0.25 ,ug/ml. Heterozygous (etgl-l/+) strains exhibited intermediate resistance (Echl phenotype) and were resistant at 1 pg/ml but sensitive at 4 ug/ml.Cloning. A plasmid (pFF119) complementing Jksl-l was selected from a yeast genomic library of strain GRF88 (13) on uracil DO medium containing FK506 at 1 pg/ml. A library of genomic DNA (provided by S. Parent) from strain YFK093 (12) was constructed as described (14) by partial Sau3A1 digestion, partial fill-in of overhangs, and insertion of the fragments into the partially filled-in Sal I site of plasmid YEp24. The YFK093 library was introduced into strain R560-1C by the spheroplast transformation method, uracil

The lipopeptide antifungal agents, echinocandins, papulacandins, and pneumocandins, kill Candida albicans by inhibiting glucan synthesis. For this fungus, there is a good correlation of in vitro enzyme inhibition with in vitro assays of MICs. Semisynthetic lipopeptides such as cilofungin, LY303366,989,560 have activity in vivo against Aspergillus infections but appear to be inactive in broth dilution in vitro tests (MICs, > 128 ,ug/ml). To understand how compounds which lack activity in vitro can have good in vivo activity, we monitored the effect of pneumocandins on the morphology ofAspergillusf migatus and A. flavus strains by light microscopy and electron microscopy and related the changes in growth to inhibition of glucan synthesis.Pneumocandin Bo caused profound changes in hyphal growth; light micrographs showed abnormally swollen germ tubes, highly branched hyphal tips, and many cells with distended balloon shapes. Aspergillus electron micrographs confirmed that lipopeptides produce changes in cell walls; drug-treated germlings showed very stubby growth with thick walls and a conspicuous dark outer layer which was much thicker in the subapical regions. The rest of the hyphal tip ultrastructure was unaffected by the drug, indicating considerable specificity for the primary target. The drug-induced growth alteration produced very compact clumps in broth dilution wells, making it possible to score the morphological effect macroscopically. The morphological changes could be assayed quantitatively by using conventional broth microdilution susceptibility assay conditions. We defined the endpoint as the lowest concentration required to produce the morphological effect and called it the minimum effective concentration to distinguish it from the no-growth endpoints used in MIC determinations.The minimum effective concentration assay was related to inhibition of glucan synthase activity in vitro and may provide a starting point for development of susceptibility testing methods for lipopeptides.Echinocandins, pneumocandins, and papulacandins are antifungal agents which inhibit the synthesis of 1,3-p-D-glucan (22,39,43,44). ,B-Glucans are vital cell wall polymers in clinically important pathogenic fungi, including Candida and Aspergillus spp. (8). The proportion of this polysaccharide in the walls of different organisms varies, but at least 10% of the dry weight of the walls of these organisms is in the form of 1,3-p-D-glucan (4). This polysaccharide is synthesized by a membrane-associated activity which has been partially characterized in yeasts such as Candida albicans and Saccharomyces cerevisiae (31,40,46). The enzyme uses UDP-glucose as a substrate and catalyzes the polymerization of a linear polymer of 1,3-p-D-glucan reported to be 60 U (40) or as much as 700 U (35) long. The Km for UDP-glucose is approximately 2 mM, and the reaction is stimulated by nucleoside triphosphates, particularly GTP (31, 42). For both C. albicans and S. cerevisiae, the in vitro product, which is synthesized without a requirement for ...

The increasing incidence of life-threatening fungal infections has driven the search for new, broad-spectrum fungicidal agents that can be used for treatment and prophylaxis in immunocompromised patients. Naturalproduct inhibitors of cell wall (

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