Mitochondrial cardiolipin sequestration of caspofungin underlies <i>Cryptococcus neoformans</i> inherent resistance and may contribute to cardiotoxicity

Cryptococcus exhibits inherent resistance to the echinocandin, caspofungin, which inhibits the synthesis of (1,3)-β-D-glucan, a key component of the polysaccharide cell wall. The essential <i>FKS1</i> gene encodes the catalytic subunit of (1,3)-β-D-glucan synthase and caspofungin effectively inhibits its activity <i>in vitro</i>, yet the drug remains ineffective against Cryptococcus, suggesting mechanisms beyond target insensitivity. The underlying mechanisms of caspofungin resistance remain unknown, although altered regulation of cell-wall remodeling genes, plasma membrane modifications, drug efflux pathways, and melanin biosynthesis have been suggested. Using boron dipyrromethene (BD-) and fluorescein (F-) labelled caspofungin, we demonstrate that caspofungin enters the cryptococcal cell and primarily accumulates in the mitochondrial inner membrane rather than the plasma membrane. We further establish that this mitochondrial accumulation is driven by a specific interaction between caspofungin and cardiolipin, a phospholipid found in mitochondrial membranes. We demonstrate that this unforeseen localization indicates that mitochondrial sequestration diminishes the effective drug concentrations at the intended target. Notably, the interaction between caspofungin and cardiolipin also takes place in human cells, establishing a mechanistic connection to caspofungin-related cardiotoxicity. Our findings reveal a previously unrecognized mechanism of echinocandin resistance in Cryptococcus and emphasize cardiolipin as an important factor in caspofungin effectiveness.