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Abstract

Cryptococcus neoformans is an opportunistic pathogenic fungus that causes meningitis and mortality in immunocompromised individuals, particularly those infected with human immunodeficiency virus (HIV) or diagnosed with acquired immunodeficiency syndrome (AIDS). Limited antifungal drug options and increasing resistance highlight the need for novel therapeutic strategies. This study first investigated the role of phosphate signaling in regulating virulence and antifungal drug tolerance for C. neoformans. We focused on the high-affinity phosphate transporters Pho84, Pho840, and Pho89, which are involved in phosphate uptake under phosphate limiting conditions. Loss of these transporters impaired phosphate acquisition, disrupted cell growth under endoplasmic reticulum (ER) stress, and altered membrane permeability and calcium sensitivity. These phenotypes were most evident in the pho840 deletion mutant. Unexpectedly, the phosphate transporter mutants had increased sensitivity to caspofungin, an echinocandin drug that is generally ineffective against C. neoformans. These mutants also showed capsule attachment defects and cell wall alterations, suggesting that phosphate deficiency compromises structural integrity to enhance drug sensitivity. Next, to test whether the caspofungin sensitivity could be induced by ER stress, we investigated the synergistic antifungal activity between the ER stress inducer tunicamycin and caspofungin. We found that the combination of these two drugs significantly reduced the minimum inhibitory concentration of caspofungin required for growth inhibition in the wild-type parental strain. This synergistic effect was associated with alterations in cell wall structure and cell membrane composition rather than an influence of capsule on drug access. The shift in cell wall composition caused by the drug combination enhanced the sensitivity of C. neoformans cells to macrophage killing. Our results further revealed additive effects between caspofungin and other ER stress inducers, further supporting the conclusion that impaired ER homeostasis influences the sensitivity of C. neoformans to antifungal drugs. Taken together, our results establish connections between phosphate signaling, ER homeostasis, and cell wall integrity in C. neoformans. Targeting the ER stress pathway combined with echinocandin treatment may be an effective strategy to overcome antifungal resistance in C. neoformans and guide future antifungal drug development.