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Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints.

ABSTRACT: A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-beta-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and K(i) increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most prominent decrease in sensitivity, while those with mutations at the N- and C-terminal ends of hot spot 1 generally retained greater sensitivity to all three drugs. Kinetic inhibitions by caspofungin, micafungin, and anidulafungin were comparable among the fks1 mutant enzymes, although absolute values did vary with specific mutations. Amino acid substitutions in Fks1p did not alter K(m) values, although some mutations decreased the V(max). Given the association of FKS1 mutations with clinical resistance, an evaluation of the kinetic parameters for the inhibition of mutant 1,3-beta-D-glucan synthase as a function of the MIC enabled an independent evaluation of the recently adopted susceptibility breakpoint for echinocandin drugs. Overall, a breakpoint MIC of >or=2 microg/ml for caspofungin captured nearly 100% of fks1 C. albicans strains when a kinetic inhibition rise threshold of or=0.5 microg/ml was required for >95% coverage of clinical isolates. However, when MIC determinations were performed in the presence of 50% serum, all fks1 mutants showed MIC values of >or=2 microg/ml for the three echinocandin drugs. The 1,3-beta-D-glucan synthase kinetic inhibition data support the proposed susceptibility breakpoint for caspofungin in C. albicans, but a lower susceptibility breakpoint (

SUBMITTER: Garcia-Effron G

PROVIDER: S-EPMC2612148 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints.

Garcia-Effron Guillermo G Park Steven S Perlin David S DS

Antimicrobial agents and chemotherapy 20081027 1

A detailed kinetic characterization of echinocandin inhibition was performed for mutant 1,3-beta-d-glucan synthase enzymes from clinical isolates of Candida albicans with nine different FKS1 mutations resulting in high MICs. Among 14 mutant Fks1p enzymes studied, the kinetic parameters 50% inhibitory concentration and K(i) increased 50-fold to several thousandfold relative to those for the wild type. Enzymes with mutations at Ser645 (S645P, S645Y, and S645F) within hot spot 1 showed the most pro ...[more]

PMID: 18955538

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Project description: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.

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Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints.

Publications

Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints.

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