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.
Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.The elongation phase of translation in fungi requires the soluble elongation factors EF1␣, EF2, and EF3. EF1␣ and EF2 are members of the GTPase superfamily of proteins and are characterized by common structural motifs and their ability to alternate between conformational states in response to binding GDP or GTP. These proteins are required for translation in all eukaryotes, while EF3 is unique to fungi and essential for fungal protein synthesis (1). EF2 catalyzes the translocation of the ribosome along messenger RNA, presumably by stimulating a gross rearrangement of the ribosome that results in peptidyl-tRNA transfer and the movement of mRNA by one codon. The protein sequence of EF2 has been highly conserved throughout evolution, with Saccharomyces cerevisiae EF2 sharing 66% identity and 85% homology to human EF2. Despite this high degree of similarity, a class of tetracyclic diterpene glycoside natural products, the sordarins, has now been identified as selective inhibitors of EF2 function in fungal protein synthesis. Sordarin, produced by species of the fungal genus Sordaria, was described as an antifungal agent in 1970 (2, 3), but the mode of action of this family has not been examined until now. In this report, we show that sordarins specifically bind to the S. cerevisiae EF2-ribosome complex and block protein synthesis by inhibiting the release of EF2 from the posttranslocational ribosome. Our observations show that it is possible to inhibit fungal EF2 specifically, which may provide an opportunity for developing antifungal agents with a unique and selective mechanism of action. EXPERIMENTAL PROCEDURESSordarin was isolated essentially as described for Sordaria arenosa (2). Reticulocyte and wheat germ lysates were obtained from Promega.Assays-IC 50 values were determined from growth inhibition assays in which cells were inoculated a...
Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3--glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of ϳ200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched ϳ300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.
A Candida krusei strain from a patient with acute myelogenous leukemia that displayed reduced susceptibility to echinocandin drugs contained a heterozygous mutation, T2080K, in FKS1. The resulting Phe655→Cys substitution altered the sensitivity of glucan synthase to echinocandin drugs, consistent with a common mechanism for echinocandin resistance in Candida spp.
The immunosuppressants FK506 and rapamycin prevent T-cell activation and also inhibit the growth of certain strains of the yeast Saccharomyces cerevisiae. It has previously been shown that yeast contains a 12-kDa cytosolic FK506-binding protein (yFKBP-12), which also possesses peptidylprolyl cis-trans isomerase activity, and thatfkbl strains lacking yFKBP-12 are resistant to rapamycin and sensitive to FK506. The absence of yFKBP-12 permitted the detection and isolation of a second FK506-and rapamycin-binding protein, which is about 13 kDa in size (yFKBP-13) and membraneassociated. Purified yFKBP-13 binds FK506 with 15-fold lower affinity than yFKBP-12 and has peptidylprolyl cis-trans isomerase activity with a similar substrate proffle. The sequence of the first 37 N-terminal amino acids was determined, and the yFKBP-13 gene (FKB2) was cloned and sequenced. A hydrophobic putative signal sequence precedes the N terminus of the mature protein. yFKBP-13 most closely resembles the membrane-associated human FKBP-13, which also possesses a signal peptide, whereas yFKBP-12 most closely resembles human FKBP-12. fkb2 and fkbl fkb2 mutants are viable and unaltered in their sensitivity to FK506, suggesting that yeast possesses an additional target for this drug. Furthermore,fkb2 null mutations confer no change in rapamycin sensitivity.These rmdings show that yFKBP-13 and yFKBP-12 have distinct functions within the cell.
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