Fungal diseases impact the lives of a millions of people across the globe, and with our current repertoire of therapeutic options dwindling, effective treatment strategies are urgently needed. Critically, the emergence of azole-resistant isolates in the clinic following prolonged treatment regimes, environmental fungicide exposure, and fungal evolution, threatens the outcome of current therapeutics, further endangering the survival of infected individuals. Here, we investigate the underpinnings of antifungal resistance using quantitative proteomics to discover protein-level signatures of fluconazole (FLC) resistance in the opportunistic human fungal pathogen, Cryptococcus neoformans. We explore ClpX, an ATP-dependent unfoldase, as a target to overcome FLC resistance and explore target efficacy through macrophage and murine models of cryptococcal infection. Here we show that disruption of ClpX, following gene deletion or targeted inhibition, re-introduces FLC susceptibility into resistant strains, rendering FLC treatment effective once again. Further, we identify and experimentally confirm mechanisms by which ClpX influences susceptibility to FLC, through association with both heme biosynthesis and ergosterol production. Overall, our results contribute to the understanding of mechanisms driving FLC resistance in a globally important fungal pathogen, and we provide avenues for targeting proteins as a therapeutic strategy to reverse antifungal resistance.

Woods, M., A. Bermas, B. Ball, B. Muselius, N. Chan, D. Gutierrez-Gongora, S. Ramezanpour, J. A. McAlister, S. A. Sieber, and J. Geddes-McAlister. "Disruption of the ATP-dependent unfoldase ClpX reverses antifungal resistance in Cryptococcus neoformans." Nature Communications 16(1), 6248 (2025).

Link: https://doi.org/10.1038/s41467-025-61412-x

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