Lem3, a phospholipid flippase subunit, influences Candida albicans pathogenicity through maintenance of plasma membrane asymmetry.

UNLABELLED: Flippases, composed of catalytic and regulatory subunits, preserve cellular integrity by maintaining the asymmetric distribution of phospholipids across the plasma membrane. Here, we provide the first functional characterization of Lem3, the regulatory subunit of the flippase complex in Candida albicans, and reveal its critical role in membrane homeostasis and pathogenesis. We demonstrate that Lem3 localizes to both the endoplasmic reticulum and plasma membrane, consistent with its role in phospholipid translocation. Phenotypic analysis of a lem3Δ/Δ mutant showed decreased susceptibility to the alkylphosphocholine analog, miltefosine. Concurrently, the mutant was defective in the inward translocation (flip) of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) across the plasma membrane, accompanied by altered levels of ergosterol and sphingolipids, indicating the requirement of Lem3 for maintaining membrane lipid composition. Notably, LEM3 deletion suppressed the enhanced phospholipid internalization seen in an rta3Δ/Δ mutant, suggesting that Rta3 negatively regulates Lem3 activity. The lem3Δ/Δ strain exhibited increased susceptibility to azole antifungals, likely due to increased membrane permeability and impaired function of the major drug efflux pump Cdr1. Strikingly, the absence of LEM3 reversed drug resistance in an azole-resistant clinical isolate, reinforcing its therapeutic relevance. Finally, Lem3 was important for biofilm formation and in vivo virulence, establishing it as a key determinant of C. albicans pathogenicity. Together, these findings position Lem3 and the flippase machinery as promising targets for antifungal therapy.

IMPORTANCE: Plasma membrane lipid composition, maintained by lipid translocators, is a key determinant of virulence in pathogenic fungi. Here, we demonstrate that Lem3, the regulatory subunit of the flippase complex, by regulating the asymmetric distribution of phospholipids, impacts the expression of virulence traits and modulates drug resistance in Candida albicans. These findings highlight how diverse cellular processes are interconnected through flippase-mediated lipid homeostasis. With the alarming rise in fungal infections and antifungal resistance, targeting lipid translocators such as Lem3 offers a promising avenue for the development of novel therapeutic targets.