Mutations in the Plasmodium falciparum Cyclic Amine Resistance Locus (PfCARL) Confer Multidrug Resistance

Abstract

Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites.

Genetic diversity of next generation antimalarial targets: A baseline for drug resistance surveillance programmes

“…The Pfatp4 and Pfcarl have been extensively studied as antimalarial targets. The Pfcarl is an uncharacterized protein-coding gene that also localises in the Golgi apparatus of the parasite and the Pfatp4 locus probably functions as a Na + -efflux ATPase (Flannery et al., 2015, LaMonte et al., 2016). Several mutations in these genes have previously been reported, particularly for Pfatp4 , to confer resistance to a growing number of antimalarial compounds that are structurally unrelated (Table 2 ) .…”

“…Here we consider eleven gene-targets of key investigated compounds that due to their efficiency might become the next antimalarial drugs, and for which mutations conferring resistance have been identified in in vitro studies (Baragaña et al., 2015, Dong et al., 2011, Flannery et al., 2015, Herman et al., 2015, Kato et al., 2016, LaMonte et al., 2016, Lim et al., 2016, McNamara et al., 2013, Ross et al., 2014). These 11 genes were also selected because they are gene-targets for a range of new antimalarial compounds already under evaluation in clinical trials.…”

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…This compound recently showed encouraging clinical efficacy, with 14 of 21 patients cured of acute uncomplicated P. falciparum malaria following treatment with a single dose 113 . Resistance to KAF156 and related imidazolopiperazines in ABS can be mediated by point mutations in the P. falciparum cyclic amine resistance locus ( pfcarl ), which encodes a conserved protein that localizes in the cis -Golgi apparatus, where it might contribute to protein sorting or membrane trafficking 111,114 . Resistance to these compounds can also be mediated by mutations in an acetyl-CoA transporter (PfACT) or a UDP-galactose transporter (PfUGT), which are localized to the endoplasmic reticulum (ER) 115 (Supplementary Table 2).…”

“…The subcellular locations of these resistance mediators suggest that imidazolopiperazines might interfere with the transport of parasite biomolecules into the ER or Golgi 115 . Parasite resistance to different chemotypes has also been associated with mutations in pfcarl , suggesting evidence of its role as a multidrug-resistance mediator rather than the actual drug target 114,116 .…”

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol

“…P . falciparum transfections have been performed with Cas9 and sgRNA either expressed on two separate plasmids or combined on one plasmid and different selectable markers have been used to maintain the plasmids in transformed parasites after transfection [22, 23, 25–27]. The selectable markers used are human dihydrofolate reductase (h dhfr ), blasticidin S deaminase ( bsd ), neomycin phosphotransferase ( neo ) and yeast cytosine deaminase/uridyl phosphoribosyl transferase (y fcu ).…”