Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes. Author summaryMiltefosine was developed as an anticancer drug but is commonly used to treat infections with the protozoan parasites Leishmania and Trypanosoma cruzi. More recently, it has been used to treat fungal infections, and in 2021 it was designated as an orphan drug by the US Food and Drug Administration for treatment of invasive candidiasis. Miltefosine is a derivative of phosphatidylcholine (PC), a major constituent of the cell membrane. PC and other phospholipids are asymmetrically distributed across the cell membrane. The mechanism of action of miltefosine is unknown. Here, we show that either increasing the activity of a putative floppase, which controls outward "flop" movement of phospholipids, or decreasing the activity of flippases, which control inward "flip" movement, results in increased resistance of the fungal pathogen Candida parapsilosis to miltefosine. This result suggests that miltefosine acts by controlling the localisation of PC or other phospholipids in the membrane. Importantly, we find that many C. parapsilosis isolates carry mutations in one flippase gene, which renders them partially resistant to miltefosine, and prone to easily acquiring increased resistance.