Poster Presentation First Malaria World Congress 2018

The natural substrates and normal physiological role of the malaria parasite’s ‘chloroquine resistance transporter’ (#339)

Sarah Shafik 1 , Simon A Cobbold 2 , Robert L Summers 1 , Alice Robert-Patterson 1 , Kawthar Barkat 1 , Simon Hogg 1 , Nicole Lancaster 1 , Sashika N Richards 1 , Manuel Llinás 3 , Adele M Lehane 1 , Malcolm J McConville 2 , Rowena E Martin 1
  1. Research School of Biology, Australian National University, Canberra, Australian Capital Territory 2601, Australia
  2. Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, VIC, Australia
  3. Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, USA

Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) play a key role in conferring resistance to antimalarial drugs, such as chloroquine. PfCRT is also essential for parasite viability; resistance-conferring mutations impart a fitness cost to the parasite and attempts to knock-out pfcrt have failed [1-3]. PfCRT is located at the membrane of the digestive vacuole, an acidic compartment in which proteases catabolise host haemoglobin and where many antimalarials accumulate, act, and/or are activated.

Previous studies have proposed that PfCRT functions as either a chloride channel, a proton pump, an activator of cation transporters/channels, or a broad-specificity transporter of organic cations – without convincing support for one claim over the others [4-6]. We have employed two experimental tools to elucidate the natural function of PfCRT – the Xenopus oocyte expression system [7-8] and isogenic parasite lines differing only in the pfcrt isoform expressed [9-10]. We found that many haemoglobin-derived peptides, varying in composition, charge, and length, inhibited [3H]chloroquine transport via mutant PfCRT isoforms. A subset of the larger peptides (4-11 residues) also trans-stimulated the PfCRT-mediated transport of [3H]chloroquine, consistent with them being substrates of the transporter. Testing of a [3H]haemoglobin-derived peptide revealed it to be a substrate of both wild-type and mutant isoforms of PfCRT, with wild-type PfCRT exhibiting the greatest level of transport activity. None of the PfCRT isoforms transported small peptides or amino acids. Moreover, we demonstrated that PfCRT transports haemoglobin-derived peptides (and several peptide mimics) in situ. However, whilst wild-type PfCRT transported a broad range of peptides, mutant PfCRT isoforms exhibited a relatively narrow specificity for peptides. Peptidomic analyses of the isogenic parasite lines confirmed that the expression of mutant PfCRT isoforms results in significant increases in the accumulation of many of PfCRT’s peptide substrates. A detailed understanding of PfCRT’s normal physiological role will be presented.

  1. Waller et al (2003) The Journal of Biological Chemistry, 278, 33593-33601.
  2. Ecker et al (2012) International Journal of Parasitology, 42, 969-974.
  3. Bushell et al (2017) Cell, 170, 260-272.
  4. Zhang et al (2002) The Journal of Biological Chemistry, 277, 49767-49775.
  5. Nessler et al (2004) The Journal of Biological Chemistry, 279, 39438-39446.
  6. Juge et al (2015) Proceedings of the National Academy of Science USA, 112, 3356-3361.
  7. Martin et al (2009) Science, 325, 1680-1682.
  8. Summers et al (2014) Proceedings of the National Academy of Science USA, 111, E1759-E1767.
  9. Sidhu et al (2002) Science, 298, 210-213.
  10. Lehane et al (2008) Journal of Cell Science, 121, 1624-1632.