P-Type ATPase Apt1 of the Fungal Pathogen Cryptococcus neoformans Is a Lipid Flippase of Broad Substrate Specificity

Abstract

Lipid flippases of the P4-ATPase family are ATP-driven transporters that translocate lipids from the exoplasmic to the cytosolic leaflet of biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the P4-ATPase Apt1p is an important regulator of polysaccharide secretion and pathogenesis, but its biochemical characterization is lacking. Phylogenetic analysis revealed that Apt1p belongs to the subclade of P4A-ATPases characterized by the common requirement for a β-subunit. Using heterologous expression in S. cerevisiae, we demonstrate that Apt1p forms a heterodimeric complex with the C. neoformans Cdc50 protein. This association is required for both localization and activity of the transporter complex. Lipid flippase activity of the heterodimeric complex was assessed by complementation tests and uptake assays employing fluorescent lipids and revealed a broad substrate specificity, including several phospholipids, the alkylphospholipid miltefosine, and the glycolipids glucosyl- and galactosylceramide. Our results suggest that transbilayer lipid transport in C. neoformans is finely regulated to promote fungal virulence, which reinforces the potential of Apt1p as a target for antifungal drug development.

Lipid transport by Candida albicans Dnf2 is required for hyphal growth and virulence

“…Human P4-ATPase mutations are linked to neurological disease (ATP8A2, ATP9A, ATP8B2, ATP10B, ATP11A), metabolic and cardiovascular disease (ATP10A, ATP10D), cholestasis and hearing loss (ATP8B1), systemic sclerosis (ATP8B4), severe Covid-19 (ATP11A), cancer progression (ATP11B) and hemolytic anemia (ATP11C) (22,23,(32)(33)(34)(35)(36)(37)(24)(25)(26)(27)(28)(29)(30)(31). The P4-ATPase Apt1-Cdc50 from Cryptococcus neoformans transports a variety of substrates, including GlcCer, and is essential in polysaccharide secretion, stress tolerance, fungal survival inside macrophages, and virulence (38)(39)(40)(41). A Candida albicans drs2∆∆ flippase mutant shows defects in hyphal growth and hypersensitivity to fluconazole (42).…”

Functional Analysis of the P-Type ATPases Apt2-4 from Cryptococcus neoformans by Heterologous Expression in Saccharomyces cerevisiae

“…Standard PCR reactions were performed with Q5 High-Fidelity DNA Polymerase (NEB, Ipswich, MA, USA). The pESC-URA vector (Agilent Technologies, Santa Clara, CA, USA) digested with BamHI was used as the backbone for APT -containing constructs without CDC50 , while the pESC-URA-APT1-GFP-CDC50-FLAG plasmid [ 19 ] digested with the same enzyme was utilized for CDC50 -containing versions. The APT2 , APT3 , and APT4 sequences were obtained as synthetic cDNA clones from Twist Bioscience (San Francisco, CA, USA).…”

“…In fact, Apt1p was recently functionally characterized based on heterologous expression in S. cerevisiae as a lipid transporter with broad substrate specificity. Besides the glycerophospholipids phosphatidylserine (PS), -choline (PC), -ethanolamine (PE), and -glycerol (PG), the transport of galactosyl ceramide (GalCer), glucosyl ceramide (GlcCer), and sphingomyelin (SM) was detected [ 19 ]. Substrate specificity and key features of Apt2-4p remain to be assessed.…”

“…Studies in vivo demonstrated that Cdc50p localizes throughout the whole secretory pathway and its deletion leads to reduced virulence, higher drug sensitivity, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane [ 9 , 17 ]. Further studies showed impaired lipid uptake at the plasma membrane via Apt1p, when expressed without Cdc50p, presumably due to impaired trafficking and therefore the retention of Apt1p in the endoplasmic reticulum [ 19 ]. Moreover, recent studies showed a strong interaction between subunits via the extracellular loop of Cdc50p, which motivated the design of antifungal peptides [ 24 ].…”

Development of Antifungal Peptides against Cryptococcus neoformans; Leveraging Knowledge about the cdc50Δ Mutant Susceptibility for Lead Compound Development

“…Recently, mutation studies discovered an essential role of Cryptococcus lipid flippase in echinocandin resistance ( 6 ). The lipid flippase is composed of the core P4 type ATPase (P4-ATPase) and a regulatory subunit Cdc50 ( 7 ). The same study found that fungal mutants with a CDC50 gene of the 250 amino acid exocytoplasmic loop region were highly sensitive to caspofungin.…”