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

Abstract

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.

Echinocandin heteroresistance causes prophylaxis failure and facilitates breakthroughCandida parapsilosisinfection

“…7a). CNV1 and CNV2 are adjacent ORFs on chromosome I, and some amplifications cover both ORFs 34 . However, CNV1 and CNV2 were evaluated independently in the RF models and were both retained by recursive feature elimination algorithm.…”

“…The complexity and diversity of C. parapsilosis genomes are in general lower than that of other Candida species, and may facilitate this proof-of-concept approach to identify heteroresistance-linked genetic features. The 118 C. parapsilosis strains in this study, although including all five clades on a phylogeny tree of 170 C. parapsilosis strains from around the world 34 , were all collected from one hospital. Future investigations that include C. parapsilosis strains with more diversified genetic backgrounds may help revise the currently selected HR features and establish an even more accurate prediction model.…”

CRISPR-Cas9 editing induces Loss of Heterozygosity in the pathogenic yeast Candida parapsilosis

“…Variants flanked by long mono/di-nucleotide repeats were removed using a custom script (https://github.com/CMOTsean/milt_variant_filtration). Heterozygous alleles with a depth ratio of below 0.25 or above 0.75 were also removed (52). To create Fig.…”

“…Annotation of gene variants and prediction of protein coding effects were performed using SIFT. A SIFT4G database for C. parapsilosis reference: CDC317 was generated for Candida parapsilosis as described in (52,56). Table S3.…”