How does pfCRT confer drug resistance to malaria?

Malaria has become resistant to treatment with drugs such as chloroquine. The honours project will examine the function of a transport protein that is a major determinant in conferring resistance to malaria.

Malaria is defined by the World Health Organisation as one of the three major causes of death world-wide. In particular, the developing world is hit hardest by the impact of this disease and treatment options are limited. Vaccine development is problematic and the use of chemotherapy remains the only therapeutic intervention. However, chemotherapy is increasingly ineffective due to the spectre of drug resistance. For example, the drug chloroquine, a mainstay of treatment for several decades, is now ineffective in most endemic malaria regions. The resistant phenotype is similar to that reported for bacterial infections, cancer chemotherapy and a host of other pathogen infections. In fact, the scientific and pharmaceutical industries appear to be locked in a battle reminiscent of the cold war era with respect to chemotherapy in a number of diseases.

Resistance to chloroquine treatment in malaria has multiple factors and includes mutations within the gene pfcrt. PfCRT encodes a protein thought to act as a drug transporter and is localised on the digestive vacuole of the malaria parasite during its erythrocytic phase. The digestive vacuole is involved in the metabolism of heme (released during digestion of haemoglobin) to non-toxic waste products. Chloroquine disrupts this process, leading to the build-up of toxic intermediates and resulting in death of the parasite. It is believed that pfCRT prevents the accumulation of chloroquine within the digestive vacuole; presumably by mediating outward transport of the drug. However, the absence of molecular data on the protein has thus far prevented the assignment of a molecular mechanism. Similarly, the endogenous role of non-mutated pfCRT remains unknown, although the suggestion of a role in glutathione transport has been mooted.

The Callaghan laboratory has recently developed a purification system for PfCRT and its subsequent reconstitution into liposomes to enable measurement of function. The project will involve the purification and reconstitution of pfCRT into liposomes, nano-discs and SMA-polymer structures to enable drug binding assays. The characteristics of chloroquine binding to pfCRT such as affinity, capacity and stoichiometry will be measured. The ability of putative inhibitors  to modulate this binding will also be assessed as will the selectivity of PfCRT to interact with uncharged, mono- or divalent charged species of chloroquine.

The project will suit students with an interest in malaria drug resistance, membrane transport processes and/or molecular studies with proteins. Training will be provided in membrane protein purification, chromatography and reconstitution into a variety of lipid systems. Further reading on the contribution of efflux pumps to drug resistance in malaria may be found in the manuscript below.

Skrzypek, R., Callaghan, R., 2017. The "pushmi-pullyu" of resistance to chloroquine in malaria. Essays Biochem 61, 167-175.