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 summary Miltefosine 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.


Introduction 43 44
Lipids in yeast plasma membranes belong to three main families: sterols, sphingolipids, and 45   Clade 2) may also share a recent origin. However, overall there is little evidence for 153 geographical clustering. Each clade includes at least one isolate from both MSK and FM (Fig  154   1). Three isolates from a clinical setting in Kuwait [46] are each located in a different clade 155 (designated by Kw in Fig 1). Interestingly, an environmental sample isolated from Irish soil, 156 UCD321, groups with clinical isolates from both MSK and FM (in Clade 5). The diversity of 157 isolates obtained from the same clinical setting, and the close relationship between isolates 158 from different geographical settings, highlight the global nature of C. parapsilosis as a 159 human pathogen. 160 161 We previously showed that the RTA3 gene, encoding a putative floppase, had undergone 162 extensive copy number amplification in 23 C. parapsilosis isolates [42]. This was also 163 observed in a small number of isolates by West et al [43]. We therefore characterized copy 164 number variants (CNVs) at the RTA3 locus in all sequenced isolates. We identified 165 increased RTA3 copy numbers in 104 of 170 isolates. Sixteen CNVs with unique endpoints 166 were observed (assigned letters A-P, Figs. 1, 2). Nine different CNVs were observed in 167 isolates from Clade 1, whereas only CNV-L is found in isolates in Clade 3, and there are no 168 CNVs in Clade 2 isolates (Fig 1). Most (15/16) of the CNVs have a single evolutionary origin, 169 and some are present in only a single isolate (Fig 1). However, CNV-A has three separate 170 origins on the tree (one in Clade 1, and two in Clade 5). 171 172 16 unique CNVs amplify RTA3 173 174 We found that RTA3 has been amplified in 16 different types of CNV, each with unique 175 endpoints. Thirteen CNV patterns result from tandem duplications that amplify the entire 176 RTA3 coding sequence (Fig 2A), and these repeat units range in size from 2.3 to 4.5 kb. 177 Some of these CNVs extend into the upstream neighbouring gene MAK16, and one (CNV-N) 178 includes the first 17 bp in the coding sequence of the downstream gene RTA2. The copy 179 number of RTA3 varies both in isolates that share the same CNV, and in isolates with 180 different CNVs (Fig 2B). Isolate MSK807 (CNV-G) has the lowest estimated copy number of 181 RTA3 among isolates with CNVs, at four copies, whereas isolate FM16 (CNV-J) has the 182 highest copy number, at 50 copies. 183

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For CNV-K, three isolates have roughly half the RTA3 copy number of other isolates with the 204 same CNV (~13x instead of ~26x; Fig 2B; S1 Table). We considered the possibility that only 205 one allele of RTA3 was amplified in these isolates (C. parapsilosis is diploid), and that both 206 alleles were amplified in other isolates. We explored this issue by using long read 207 sequencing (Oxford Nanopore) of C. parapsilosis MSK812 which is one of the CNV-K 208 isolates with fewer copies. We found that it has 8 copies of RTA3 at one allele and 6 copies 209 at the other (S1A Fig). Similarly, sequencing of isolate MSK478 (which also has CNV-K, with 210 higher copy number of RTA3) showed that it has at least 11 copies at both alleles (S1B Fig). Therefore the variation in copy number among isolates with CNV-K is due to expansion or 212 contraction of the array in both alleles, and not due to hemizygosity for the amplification. gene RTA2 (Fig 2A,C). This repeat structure generates an array of in-frame RTA2/RTA3 217 gene fusions, with the N-terminus derived from RTA2 and the C-terminus from RTA3 (Fig  218   2C). RTA3 and RTA2 are very similar genes, which likely facilitated the fusion event. In 219 CNV-H, the Rta3 protein is slightly truncated because this CNV consists of a tandem 220 duplication with an endpoint 31 bp upstream of the stop codon of RTA3, resulting in a protein 221 that is 10 amino acids shorter than the wild type. fusion of the CoRTA2 and CoRTA3 genes, similar to CNV-P in C. parapsilosis (Fig 2D). 247 Interestingly, the final C. orthopsilosis CNV (CNV-Co4) involves a deletion. In the two strains 248 containing CNV-Co4, the 3' end of CoRTA3, the 5' end of CoRTA2, and the intergenic space 249 between them are deleted at one allele ( Fig 2E). This results in a new fusion gene, with the 250 N-terminus derived from CoRTA3 and the C-terminus derived from CoRTA2. Single copies 251 of CoRTA2 and CoRTA3 are intact on the other allele ( Fig 2E). This is the only example we to at least 30 µg/ml, as can isolates with CNV-B (which amplifies only the region upstream of 263 RTA3). The CNV with the weakest effect on MF resistance is CNV-G, which can tolerate 10-264 15 μg/ml, which is still higher than the reference strain CLIB214. 265 When different isolates carrying the same CNV are compared, miltefosine resistance is seen 266 to be correlated with the copy number of RTA3 ( Fig 3B).

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The isolates have variable levels of sensitivity to fluconazole (Fig 3), but the copy number of 287 RTA3 does not correlate with susceptibility to this drug. For example, isolates MSK815 and 288 UCD321 which have 17 and 27 copies of CNV-D differ in their susceptibility to miltefosine, 289 but they both tolerate fluconazole levels of 6 µg/ml. 290 We also found that deleting RTA3 in the reference isolate of C. parapsilosis (CLIB214) by 291 CRISPR-Cas9 editing [53] results in increased sensitivity to miltefosine ( Fig 3C) affected by deleting RTA3 (Fig 3C). 295 Increasing the RTA3 copy number correlates with increased expression, as shown by West only the promoter region is amplified (CNV-B). We found that RTA3 expression is 301 approximately 22-fold higher in C. parapsilosis MSK808, and 2.8-to 6.6-fold higher in C. 302 parapsilosis MSK802 and MSK803 than in the reference strain CLIB214 (Table 1)

Generation of miltefosine-resistant strains by experimental evolution 308
The prevalence of RTA3 amplification in C. parapsilosis isolates suggests that there may be 309 some strong selective pressure inducing amplification. To determine if miltefosine is the 310 driving force, we characterized the effect of exposing isolates to increasing concentrations of 311 miltefosine, in an adaptive laboratory evolution approach. We started with two isolates with 312 only one copy of RTA3 at each allele that were in the same clade as other isolates that had 313 undergone amplification -C. parapsilosis MSK795 from Clade 1, which is related to isolates 314 that have undergone 4 different CNVs (B, C, E, and F), and C. parapsilosis MSK247 from 315 Clade 5, which is related to isolates with CNVs A, D, J and M (Fig 1; Fig 4). Five lineages 316 were evolved from MSK247 (247A to 247E) and one from MSK795 (795B). Isolates were 317 cultured in YPD with increasing concentrations of miltefosine, up to a maximum of 32 µg/ml, 318 over a 26-day period (Fig 4A). Sixteen randomly picked evolved colonies from each lineage 319 tolerated miltefosine levels of 40 µg/ml (Fig 4B). The genomes of ten isolates derived from 320 MSK247 and two from MSK795 were sequenced, together with the parental strains. The

Miltefosine-resistant isolates acquired homozygous disruptions in two flippase genes 341
We did not find any evidence of amplification of the RTA3 locus in any of the evolved 342 miltefosine-resistant strains. However, by comparing the sequences of the evolved strains to 343 those of the parental strains, we identified two genes with homozygous loss-of-function 344 variants in all 10 resistant isolates: CPAR2_102700 and CPAR2_303950. These variants 345 include frameshifts, nonsense mutations, and missense mutations that are predicted to be 346 deleterious (Fig 5A). CPAR2_102700 and CPAR2_303950 encode putative Class 3 P4-347 ATPases and are homologs of the PC flippase genes DNF1 and DNF2 in S. cerevisiae (Fig  348   5B). 349 The two sequenced isolates derived from C. parapsilosis MSK795 (795B1 and 795B16) 350 acquired mutations in both CPAR2_102700 and CPAR2_303950, whereas lineages derived 351 from C. parapsilosis MSK247 acquired mutations only in CPAR2_303950 (Fig 5A). However, 352 subsequent analysis revealed that C. parapsilosis MSK247 contains a homozygous natural 353 variant in CPAR2_102700, resulting in a Trp-to-Stop nonsense mutation (W1280X; Fig 5A).
Deleting CPAR2_102700 and CPAR2_303950 increases resistance to miltefosine, but not to 375 fluconazole. CPAR2_102700 and CPAR2_303950 were deleted singly or together in the C.

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This result suggests that maximum levels of miltefosine resistance requires inactivation of 380 both genes, CPAR2_30395 and CPAR2_102700. To test this hypothesis, we deleted them, 381 both separately and together, in the C. parapsilosis CLIB214 genetic background using 382 CRISPR-Cas9 editing (Fig 5C). Deleting CPAR2_102700 alone in C. parapsilosis CLIB214 383 allows growth up to 5 µg/ml miltefosine, whereas deleting CPAR2_303950 alone has no 384 effect ( Fig 5C). However, strains in which both CPAR2_303950 and CPAR2_102700 are 385 deleted tolerate at least 30 µg/ml miltefosine, a similar concentration to strains derived from 386 the experimentally evolved isolates (MSK795B1, MSK247C1) (Fig 5C). Deleting 387 CPAR2_102700 alone, or in combination with CPAR2_303950, slightly increases sensitivity 388 to fluconazole (Fig 5C).  possible that a rarely used origin is present. It is likely that the amplified region contains a 455 promoter because expression of RTA3 is increased (Table 1). 456 Our results strongly suggest that there is selective pressure driving amplification of RTA3 in 457 C. parapsilosis. This conclusion is based on our observation that amplification occurred in 458 isolates from diverse genetic and environmental backgrounds, distributed across the C. 459 parapsilosis phylogeny (Fig 1). We also found that there have been multiple independent were then diluted to an A600 = 0.1, incubated for 10 h, and the miltefosine concentration was 518 doubled. The 5-day cycle was repeated until a concentration of 32 µg/ml of miltefosine was 519 reached (see Fig 4A) France were screened for resistance to miltefosine, and 16 isolates were chosen for 528 sequencing. Genomic DNA was isolated from 5 ml overnight cultures in YPD at 30℃ using Oxford Nanopore sequencing. Strains were grown overnight in 50 ml YPD broth and 546 genomic DNA was extracted from approximately 4x10 9 cells using a QIAGEN Genomic-tip 547 100/G kit (10223, QIAGEN) with minor modifications. The lyticase incubation was extended 548 to 2 h, and the proteinase K incubation was extended to overnight (~15 h). DNA libraries 549 were prepared using three different kits as per manufacturer's instructions. Libraries from C. 550 parapsilosis MSK812 and UCD321 were prepared using a Ligation Sequencing Kit (SQK-551 LSK109, Oxford Nanopore) using 1 µg of DNA per strain. DNA was repaired using a 552  and MarkDuplicates tools respectively [44] . Variants were called with GATK HaplotypeCaller 580 using the tag "--genotyping_mode DISCOVERY", combined using GATK CombineGVCFs 581 and joint-genotyped using GATK GenotypeGVCFs. Variant files were filtered for read depth 582 (<15) using and genotype quality (<40) using GATK VariantFiltration. Additionally, clusters of 583 SNPs (5 SNPs in a 100bp window) were filtered using GATK VariantFiltration. A custom 584 script was used to remove variants that were flanked on either side by a long string of mono-585 or dinucleotide repeats, and variants that were called as heterozygous but had an allele 586 depth ratio <0.25 or >0.75 (https://github.com/CMOTsean/milt_variant_filtration). 587 Additionally, for tree construction, indels were excluded using GATK SelectVariants with the 588 tag "--select-type-to-include SNP". For analysis of the evolved strains, a custom script was 589 used to filter out variants in the evolved strains that were also present in the respective 590 parent strain (https://github.com/CMOTsean/milt_variant_filtration). 591 592 SIFT4G analysis. A SIFT prediction database was created for C. parapsilosis using the 593 SIFT4G algorithm [71], and the recommended Uniref90 database as a reference for protein sequences [72]. The C. parapsilosis prediction database was used to annotate variants from 595 the evolved strains with whether they are likely to be deleterious to protein function. 596 For each annotated gene in C. parapsilosis, the number of evolved strains which carried a 597 variant predicted to be protein function-affecting by SIFT in that gene was tallied. Variants 598 were also visualised using Integrative Genomics Viewer (IGV) to manually check results 599