Assessing Resistance to the Echinocandin Antifungal Drug Caspofungin in Candida albicans by Profiling Mutations in FKS1

Abstract

Resistance of clinical isolates of Candida albicans to the echinocandin drug caspofungin is slowly emerging and is linked to mutations in short conserved regions in the FKS1 gene. The most prominent changes occurred at the serine 645 position in Fks1p with substitutions of proline, tyrosine, and phenylalanine. An allele-specific real-time PCR molecular-beacon assay was developed for rapid identification of drug resistance by targeting FKS1 mutations. Mutations altering serine 645 were reliably identified in both heterozygous and homozygous states. The molecular-beacon assay was used to evaluate two large collections of spontaneous mutants from separate strains of C. albicans with resistance (MICs, >16 g/ml) to caspofungin with the goal of understanding the relationship between FKS1 mutations and echinocandin resistance. Of 85 resistant isolates recovered, all were identified with mutations in FKS1; 93% showed changes at Ser645, with 62% displaying a characteristic S645P substitution expressed as either a homozygous or a heterozygous mutation in FKS1. Two other prominent amino acid substitutions, S645Y and S645F, were found at frequencies of 22% and 8%, respectively. Three new mutations were also identified: T1922C, G1932T, and C1934G, encoding F641S, L644F, and S645C substitutions, respectively. One strain had the double amino acid substitution L644F and S645C. Allele-specific probes were combined in a multiplex assay for reliable screening of known FKS1 mutations. These data support the importance of FKS1p substitutions in echinocandin resistance and demonstrate the feasibility of applying molecular screening for routine resistance assessment.The echinocandin drugs, caspofungin, micafungin, and anidulafungin, are the first of a new class of antifungal compounds that target the fungal cell wall by blocking ␤-1,3-glucan synthase (4, 6, 9). These drugs have broad-spectrum activity against Candida and Aspergillus spp. without cross-resistance to existing agents and therefore are effective against azole-resistant yeasts and molds (1,7,15,18,19). Importantly, due to their critical effect on the cell wall, echinocandins kill yeasts (5). Use of caspofungin (CANCIDAS), the first approved echinocandin, in the clinic is expanding rapidly, and it is now widely used along with triazole drugs, such as voriconazole, for primary therapy against yeast and molds. The entry of the closely related drugs micafungin and anidulafungin will further extend the clinical scope of this highly efficacious class of drugs. As patient exposure to caspofungin grows, and as micafungin and anidulafungin augment the market, it is anticipated that the number of clinical isolates with highly elevated MICs will increase. Because the echinocandins are the first new major antifungal drug class to enter the market in decades, it is vital to assess the nature of developing resistance mechanisms to this class of drugs.Recently, we reported that specific mutations in two highly conserved regions of the Fks1p subunit of glucan synthase, a putative large po...

Resistance to echinocandin-class antifungal drugs

“…Mutations at the C-terminal end of HS1 show the weakest phenotypes (caspofungin MIC =2 μg/ml). Despite the wide range of mutations, the highest frequency of substitutions were found at Ser645 (S645P, S645F or S645Y) within HS1 Balashov et al, 2006). The mutations are dominant, displaying phenotypic resistance when present in both homozygous and heterozygous forms in C. albicans.…”

“…A comparable mutation in C. albicans Fks1, P649C, confers an equivalent level of reduced susceptibility . C. orthopsilosis and C. metapsilosis, which are closely-related to C. parapsilosis (Tavanti et al, 2005), contain the P649A polymorphism and show reduced susceptibility (Balashov et al, 2006). Furthermore, C. orthopsilosis has an additional I1359V polymorphism in HS2, resulting in even slightly higher MIC values .…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…Although the echinocandins have been used in the clinic for only a relatively short time, cases of resistant isolates from patients treated with this antifungal have been documented (37,224,309,447). The echinocandins are large lipopeptide molecules that act as noncompetitive inhibitors of (1,3)-␤-Dglucan synthase, although the mechanistic details remain enigmatic (120,143).…”

Plant-derived antifungal agent poacic acid targets β-1,3-glucan

“…Given that poacic acid may directly target the cell wall or the integrity signaling pathway, we tested whether the mode of action of poacic acid differed from that of the echinocandin caspofungin. Echinocandins damage the yeast cell wall by noncompetitive binding of the β-1,3-glucan synthase complex at the Fks1p subunit (41,42). Synergistic interactions occur with drugs targeting the same or a functionally related pathway but through different targets (43).…”