A Putative P-Type ATPase, Apt1, Is Involved in Stress Tolerance and Virulence in Cryptococcus neoformans

The export of virulence factors, such as the capsule polysaccharide, to the cell surface is a critical aspect of the pathogenicity ofCryptococcus neoformans. A view of capsule export via exocytosis and extracellular vesicles is emerging, but the molecular mechanisms underlying virulence factor transport pathways remain to be established. In this study, we characterized theAPT1 gene, which encodes a predicted integral membrane P-type ATPase belonging to the type IV, Drs2 family of aminophospholipid translocases (flippases) (APTs). APTs maintain the phospholipid asymmetry that is critical in membrane fusion events for trafficking and in establishing cell polarity. Deletion of theAPT1 gene resulted in phenotypes consistent with similar roles inC. neoformans . These included altered actin distribution, increased sensitivity to stress conditions (oxidative and nitrosative stress) and to trafficking inhibitors, such as brefeldin A and monensin, a reduction in exported acid phosphatase activity, and hypersensitivity to the antifungal drugs amphotericin B, fluconazole, and cinnamycin. However, there was no difference in growth, capsule size, or melanin production between the wild type and theapt1 mutant strains at either 30°C or 37°C. Despite the absence of an influence on these major virulence factors, Apt1 was required for survival during interactions with macrophages, andapt1 mutants exhibited attenuated virulence in a mouse inhalation model of cryptococcosis. Therefore, Apt1 contributes to virulence and the stress response inC. neoformans through apparent functions in membrane fusion and trafficking that do not influence the deposition of major virulence factors, such as capsule and melanin, outside the cell.