Peroxin Pex8 couples stress responses, antifungal tolerance, and virulence regulation in <i>Candida albicans</i>

<i>Candida albicans</i>, a World Health Organization critical-priority fungal pathogen, represents the predominant cause of candidemia. Therapeutic failure is sometimes driven by antifungal tolerance, a phenotype distinct from resistance, whose underlying mechanisms remain incompletely defined. Here, we identify the peroxisomal protein Pex8 as a key regulator of tolerance to both azoles and amphotericin B. Although neither deletion nor overexpression of <i>PEX8</i> altered minimum inhibitory concentrations, both modifications significantly reduced drug tolerance, as demonstrated by reduced fractional growth (FoG₂₀) and impaired survival under drug pressure. RNA-seq analysis showed that <i>PEX8</i> overexpression leads to downregulation of ergosterol biosynthesis genes and remodeling of stress-response pathways, suggesting a possible transcriptional basis for the loss of azole tolerance. Complementary lipidomic profiling demonstrated that <i>PEX8</i> genetic manipulations induce extensive membrane lipid remodeling, characterized by specific alterations in ceramide and lysophospholipid subclasses, thereby revealing an ergosterol-independent mechanism underlying amphotericin B tolerance attenuation. Phenotypically, <i>PEX8</i> overexpression attenuated serum-induced hyphal morphogenesis and reduced virulence in a <i>Galleria mellonella</i> infection model, consistent with downregulation of hyphal-associated and virulence-related genes. Our findings establish Pex8 as a central coordinator of oxidative stress adaptation, membrane homeostasis, filamentation, and pathogenicity, revealing a promising target for combating antifungal tolerance.