Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling

Abstract

Expanding echinocandin use to prevent or treat invasive fungal infections has led to an increase in the number of breakthrough infections due to resistant Candida species. Although it is uncommon, echinocandin resistance is well documented for Candida albicans, which is among the most prevalent bloodstream organisms. A better understanding is needed to assess the cellular factors that promote tolerance and predispose infecting cells to clinical breakthrough. We previously showed that some mutants that were adapted to growth in the presence of toxic sorbose due to loss of one chromosome 5 (Ch5) also became more tolerant to caspofungin. We found here, following direct selection of mutants on caspofungin, that tolerance can be conferred by at least three mechanisms: (i) monosomy of Ch5, (ii) combined monosomy of the left arm and trisomy of the right arm of Ch5, and (iii) an aneuploidyindependent mechanism. Tolerant mutants possessed cell walls with elevated chitin and showed downregulation of genes involved in cell wall biosynthesis, namely, FKS, located outside Ch5, and CHT2, located on Ch5, irrespective of Ch5 ploidy. Also irrespective of Ch5 ploidy, the CNB1 and MID1 genes on Ch5, which are involved in the calcineurin signaling pathway, were expressed at the diploid level. Thus, multiple mechanisms can affect the relative expression of the aforementioned genes, controlling them in similar ways. Although breakthrough mutations in two specific regions of FKS1 have previously been associated with caspofungin resistance, we found mechanisms of caspofungin tolerance that are independent of FKS1 and thus represent an earlier event in resistance development.

Genomics‐driven discovery of a novel self‐resistance mechanism in the echinocandin‐producing fungus Pezicula radicicola

“…A compensatory increase in chitin synthesis also has been linked to increased resistance to echinocandin in Candida and Aspergillus strains including the echinocandin B‐producing fungus Aspergillus pachycristatus ATCC 58397 (NRRL 11440) (Walker et al ., ; Toth et al ., ). Finally, aneuploidy in chromosome 5 of C. albicans and downregulation of genes involved in cell wall biosynthesis may be involved in some forms of echinocandin resistance (Yang et al ., ). Varying degrees of echinocandin resistance and sensitivity in Aspergillus species have described previously.…”

Elevation of cell wall chitin via Ca2+–calcineurin‐mediated PKC signaling pathway maintains the viability of Candida albicans in the absence of β‐1,6‐glucan synthesis

“…A salvage septum synthesized by other redundant chitin synthases can compensate for the loss of CHS1 , which enables the cell division to proceed (Walker et al , ). Furthermore, stimulation of chitin synthesis can offset the loss of β‐1,3‐glucan caused by the echinocandin treatment (Walker et al , ; Yang et al , ). Moreover, we revealed previously that in addition to the chitin and β‐1,3‐glucan synthesis machinery, β‐1,6‐glucan synthesis also possesses a compensatory strategy in C. albicans (Han et al , ).…”

Candida albicans exhibits distinct cytoprotective responses to anti-fungal drugs that facilitate the evolution of drug resistance

“…We chose a concentration of 0.01 mg/ml for both fluconazole (FCZ) and nystatin (NYS), representing a moderate dosage relative to their MIC 50 s 8,10,48-50 . For caspofungin (CSP), a compound that interrupts cell wall biosynthesis [51][52][53] , a concentration of 1 nanogram/ml. This is well below its MIC 50 levels and chosen in order to increase the number of survivors of this compound ( Methods 3 ).…”

“…A concentration of 0.01 mg/ml was chosen for both fluconazole (Sigma #F8929) and nystatin (Sigma #N6261), representing a moderate dosage relative to their reported MIC50 levels 8,10,[48][49][50] . A concentration of 1ng/ml was used for caspofungin (Sigma #SML0425), a compound that interrupts cell way biosynthesis [51][52][53] ; this is well below its reported MIC50 levels and chosen in order to ensure a sufficient number of survivors to generate single cell profiles. The target drug was delivered to the individual colonies from step 2 and incubated at 30°C for 48 or 72 hours.…”