Fungal echinocandin resistance

Abstract

The echinocandins are the newest class of antifungal agents in the clinical armory. These secondary metabolites are non-competitive inhibitors of the synthesis of beta-(1,3)-glucan, a major structural component of the fungal cell wall. Recent work has shown that spontaneous mutations can arise in two hot spot regions of Fks1 the target protein of echinocandins that reduce the enzyme's sensitivity to the drug. However, other strains have been isolated in which the sequence of FKS1 is unaltered yet the fungus has decreased sensitivity to echinocandins. In addition it has been shown that echinocandin-treatment can induce cell wall salvage mechanisms that result in the compensatory upregulation of chitin synthesis in the cell wall. This salvage mechanism strengthens cell walls damaged by exposure to echinocandins. Therefore, fungal resistance to echinocandins can arise due to the selection of either stable mutational or reversible physiological alterations that decrease susceptibility to these antifungal agents.

Fig. 1

Signalling pathways that regulate cell wall remodelling of Candida albicans. The HOG1, CEK1 and PKC MAP kinase cascades and the Ca²⁺–calcineurin signalling pathway control a number of cellular processes including cell wall synthesis and maintenance. Upstream of the MAP kinase cascades are membrane sensors (Wsc family, Mid2, Mtl1, Sho1 and Sln1) that detect alterations in the wall and convey the signal to the internal components of the pathway. The PKC pathway plays a critical role in the response to echinocandins and the first component that is activated is Rho1, which also acts as a regulatory sub-unit of β-(1,3)-glucan synthase. Rho1 activates Protein kinase C, which phosphorylates and activates the MAP kinase kinase kinase Bck1, which in turn activates the MAP kinase kinase Mkk2, which then phosphorylates Mkc1. A number of transcription factors contribute to the response to echinocandins including Cas5 and Sko1. The Rlm1 and Bcr1 transcription factors also control the expression of a number of cell wall related genes. In S. cerevisiae Pkc1 is involved in targeting Chs3 to the plasma membrane in response to heat shock. Significant re-wiring of signalling pathways is evident in C. albicans, compared to the S. cerevisiae paradigm, for example, the role of the CaSko1 transcription factor in response to caspofungin is independent of Hog1 MAP kinase but involves the Psk1 PAK kinase. The calcineurin pathway is activated by calcium that may enter cells through membrane-localised channels Cch1 and Mid1 or a third minor channel Fig. 1, alternatively calcium may be released from intracellular stores. Ca²⁺ binds to and activates calmodulin (Cmd1) that in turn activates the phosphatase calcineurin, which is made up of two sub-units Cna1 and Cnb1. Calcineurin dephosphorylates the transcription factor Crz1, which moves into the nucleus and induces expression of genes through binding to CDREs (calcium dependent response elements) within their promoter sequences. FK506 binding to Fpr1 or cylosporin A binding to cycophilin Cpr1 results in calcineurin inhibition. Adapted from (Levin, 2005; Steinbach et al., 2007b).

Fig. 2

Treatment of fungal cells with caspofungin. Transmission electron micrographs of Candida albicans yeast cells grown in YPD medium at 30 °C for 6 h in the absence (a) or presence of 0.032 μg/ml caspofungin (b) and (c). Scale bars represent 2 μm in (a) and (b) and 1 μm in (c). Light microscopy showing a Aspergillus fumigatus hypha after 13 h (d) and 14 h (e) treatment with 2 μg/ml caspofungin, a lysed tip is marked by the arrow. In (d) and (e) scale bar is equal to 10 μm.

Fig. 3

Treatment with caspofungin increases chitin content in C. albicans. (A) Transmission electron micrographs showing WGA-colloidal gold staining of chitin in wild-type C. albicans (i) and after treatment with a sub-MIC concentration (0.032 μg/ml) of caspofungin (ii). (B) CFW staining was used to assess chitin levels of cells grown in YPD alone (i), after treatment with 0.032 μg/ml caspofungin (ii), in YPD with 200 mM CaCl₂ and 100 μg/ml CFW (iii) and after pre-growth with CaCl₂ and CFW followed by exposure to 0.032 μg/ml caspofungin (iv). DIC images (top panels) and CFW fluorescent images (bottom panels). Scale bars are (A) 0.2 μm and (B) 2 μm.