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Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

ABSTRACT: A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and was able to detect all homozygous mutants included in the collection of strains, demonstrating that it is a robust, quick, and labor-saving method that is suitable for a routine clinical diagnostic laboratory.

SUBMITTER: Dudiuk C

PROVIDER: S-EPMC4473184 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

Dudiuk Catiana C Gamarra Soledad S Jimenez-Ortigosa Cristina C Leonardelli Florencia F Macedo Daiana D Perlin David S DS Garcia-Effron Guillermo G

Journal of clinical microbiology 20150415 7

A rapid molecular-based assay for the detection of the Candida albicans FKS1 gene mutations responsible for resistance to echinocandin drugs was designed and evaluated. The assay consisted of a multiplexed PCR set of 5 tubes able to detect the most commonly described resistance mechanism, including FKS1 hot spot 1 and hot spot 2 mutations. The performance and specificity of the assay was evaluated using a double-blinded panel of 50 C. albicans strains. The assay showed a sensitivity of 96% and w ...[more]

PMID: 25878347

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Project description: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=50-fold for the K(i) was used as a measure of susceptibility. A similar MIC breakpoint for micafungin and anidulafungin was less inclusive, and a projected MIC 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 (<or=0.5 microg/ml) may be more appropriate for anidulafungin and micafungin. Overall, the data indicate that MIC testing with caspofungin may serve as a surrogate marker for resistance among the class of echinocandin drugs.

Project description:Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.

Dataset Information

Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

Publications

Quick Detection of FKS1 Mutations Responsible for Clinical Echinocandin Resistance in Candida albicans.

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