Regulation of Oxidative Stress Associated with an SSK2 Point Mutation Promotes Amphotericin B Resistance in Cryptococcus gattii.
Deng Heng Yu HY, Zang Xue Feng XF, Zhao En Qi EQ, Huang Ye Mei YM et al.
Cryptococcus gattii ( C. gattii) is a pathogenic respiratory fungus that rapidly disseminates through the central nervous system. The current clinical treatments for C. gattii infections are limited by prolonged therapeutic courses, high drug dosages, and insufficient antifungal options. This study aimed to explore the molecular mechanisms underlying amphotericin B (AmB) resistance in C. gattii and provide novel insights for developing targeted antifungal strategies. A total of 27 clinical C. gattii isolates were subjected to antifungal susceptibility testing, multilocus sequence typing (MLST) for phylogenetic analysis and resistance-related mutation screening. The CRISPR/Cas9 system was used to construct an SSK2-knockout mutant ( ssk2Δ-G4). Capsule and melanin assays, murine pulmonary infection models, and transcriptomic analyses were performed to verify gene function, evaluate therapeutic efficacy, and explore the underlying regulatory mechanisms. G4 isolates exhibited prominent AmB resistance with a novel SSK2 c.793A>G missense mutation (p.Thr265Ala; hereafter SSK2 T265A). SSK2 knockout significantly enhanced AmB susceptibility, whereas SSK2 T265A complementation partially restored the resistant phenotype. AmB treatment markedly reduced pulmonary fungal burden and inflammatory infiltration ( P < 0.05) and improved survival ( P < 0.001) in ssk2Δ-G4-infected mice. Mechanistically, this mutation upregulated CAT1 expression and enhanced the reactive oxygen species-scavenging capacity and oxidative stress tolerance. SSK2 c.793A>G is closely associated with AmB resistance and functionally contributes to the resistance phenotype. SSK2 could serve as a potential therapeutic target for developing AmB synergistic sensitizers and as a research direction for future synergistic treatments of clinically resistant C. gattii strains.