Resistance to echinocandin-class antifungal drugs

Abstract

Invasive fungal infections cause morbidity and mortality in severely ill patients, and limited drug classes restrict treatment choices. The echinocandin drugs are the first new class of antifungal compounds that target the fungal cell wall by blocking beta-1,3-d-glucan synthase. Elevated MIC values with occasional treatment failure have been reported for strains of Candida. Yet, an uncertain correlation exists between clinical failure and elevated MIC values for the echinocandin drugs. Fungi display several adaptive physiological mechanisms that result in elevated MIC values. However, resistance to echinocandin drugs among clinical isolates is associated with amino acid substitutions in two "hot-spot" regions of Fks1, the major subunit of glucan synthase. The mutations, yielding highly elevated MIC values, are genetically dominant and confer cross-resistance to all echinocandin drugs. Prominent Fks1 mutations decrease the sensitivity of glucan synthase for drug by 1000-fold or more, and strains harboring such mutations may require a concomitant increase in drug to reduce fungal organ burdens in animal infection models. The Fks1-mediated resistance mechanism is conserved in a wide variety of Candida spp. and can account for intrinsic reduced susceptibility of certain species. Fks1 mutations confer resistance in both yeasts and moulds suggesting that this mechanism is pervasive in the fungal kingdom.

Fig. 1. Schematic diagram depicting interactions between cell wall biosynthesis and cell integrity/stress pathways in yeasts

Glucan synthase (GS), comprised of catalytic subunit Fks1 (or Fks2) and its activator Rho1, is responsible for synthesis of the principal cell wall component β-1,3-glucan. It is the target of inhibition for echinocandin drugs. The GTP binding protein Rho1 plays a central role in cell wall biosynthesis and regulation through a direct activation of GS. It also mediates a wide range of cellular responses through Pkc1 and the protein kinase C (PKC) cell integrity pathway, as well as through the secretary (Sec3) pathway. External stimuli, including cell wall stress due to echinocandin action, are mediated in part through receptors Wsc1 and Mid2, which interact with Rho1 leading to a range of secondary interactions. The responses include activation of the PKC cell integrity pathway and Slt2, as well as modulation of chitin synthase genes Chs1/2/3/8. Feedback of the cell integrity pathway to cell wall biosynthesis and modeling also involves transcription factor Rlm1, which regulates genes encoding enzymes involved in cell wall biosynthesis. Ca²⁺-calcineurin indirectly affects Fks1 through Crz1. Overall, these complex pathways allow the cells to adapt to echinocandin action and the ensuing cell wall stress via compensatory cell wall biosynthesis and remodelling. (Schematic adapted from Lesage and Bussey, 2006; Selvaggini et al., 2004; Hahn and Thiele, 2002.)

Fig. 2. Amino acid alignment of Fks “hot-spot” 1 regions from 11 different fungi

The dark shading represents identical amino acids, while the gray shading shows homologous residues. NCBI accession numbers for FKS genes were as follows: D88815 (Ca), XM 446406 (Cg), AF159533 (Cp), AB091349 (Cn v.n), U08459 (Sc), AF198090 (Cl), DQ017894 (Ck), AF148715 (Pb), D78352 (Sp), U51272 (An), and U79728 (Af).

Fig. 3. Summary of resistance properties associated with clinical isolates of C. albicans from a single patient

Clinical isolates obtained from a single patient were evaluated for MIC values according to CLSI protocol M27A2, mutations in HS1 and HS2, IC50 values for inhibition of glucan synthase, and ED90 values for reduction of kidney burdens in a murine candidiasis model. (Adapted from Park et al., 2005.)

Fig. 4. Inhibition of glucan synthase from fks1 mutant strains by caspofungin

GS activity was assessed by the incorporation of [³H]-glucose into radiolabelled product. (A) Caspofungin inhibition of wild type (squares) and a homozygous S645Y/S645Y strain (triangles). (B) Caspofungin titration curves for the C. albicans wild type (squares), a heterozygous S645P/WT strain (triangles) and homozygotic pseudo-haploid S645P/null strain (circles). (Adapted from Park et al., 2005)

Fig. 5. Summary of hot-spot 1 and 2 substitutions associated with resistance in diverse Candida spp

Amino acid substitutions in HS1 and HS2 were evaluated following DNA sequence analysis FKS1 genes from susceptible and resistant Candida spp. (Park et al. 2005). MIC values were determined according to CLSI protocol M27A2. Mutations yielding resistant strains (caspofungin MIC>2 μg/ml) are shown in red; those producing weak resistance (caspofungin MIC =1–2 μg/ml) are shown in yellow, and mutations with no resistance phenotype are shown in green.