Primers, plasmids and strains used in this study.

Antibiotic tolerance, by which susceptible bacteria survive at high bactericide doses, is known to cause treatment failure in clinical practice. However, the impact of antifungal tolerance on clinical outcomes remains poorly understood. Here, we observed that candidemia cases caused by echinocandin-tolerant Candida tropicalis exhibited higher mortality rates during caspofungin treatment by conducting a comprehensive seven-year retrospective analysis. C. tropicalis develops tolerance to caspofungin by forming multicellular aggregates, a process linked to defects in cell division, both in vitro and in vivo. Our omics-based profiling results reveal that C. tropicalis develops tolerance through the intricate modulation of cell wall integrity and cell division pathways, particularly through the activation of chitin synthesis and the downregulation of cell division-related genes. The overexpression of cell division-related factor Ace2 can suppress the tolerance of C. tropicalis to caspofungin by delaying the formation of multicellular aggregates. Moreover, calcineurin inhibitors can suppress the tolerance of C. tropicalis by disrupting these adaptive molecular changes, thereby significantly enhancing the antifungal efficacy of caspofungin in a Galleria mellonella model. Collectively, our findings provide evidence that C. tropicalis acquires echinocandin tolerance through morphological alterations, and that inhibiting calcineurin may be a promising method to reduce this tolerance.