Parasite Genetics and Adaption - Project B2

'New Permeability Pathway' structure and function in Plasmodium falciparum

Host lab: Melanie Rug (ANU)

HUB partner: Andreas Hermann and Thomas Korte

ANU partner: Alexander Maier and Kevin Saliba

   (click to enlarge images)

Modified from Gupta et al. Drug Resist Updat 2015
Modified from Gupta et al. Drug Resist Updat 2015
Counihan et al., eLife 2017
Counihan et al., eLife 2017

The asexual stages of Plasmodium falciparum, the deadliest of malaria parasites, reside in human erythrocytes to evade the host’s immune system. In order to survive in a cell that is devoid of protein synthesis and trafficking machinery, it deploys ‘New Permeability Pathways’ (NPP) for nutrient uptake and toxic waste disposal. Many physiological and pharmacological studies have been undertaken to functionally characterise host cell surface transporters involved in this pathway, but information on their molecular characterisation is still scarce. Plasmodial surface anion channel (PSAC), of yet unknown molecular nature, has been described to mediate uptake of a range of ions and solutes between host cell surface and parasite and is known drug target (1).

 

Most physiological assays are undertaken on whole cell systems where the complexity of multiple surface transporters on the cell surface and the presence of intracellular enzyme pathways that can metabolise the substrates used for measuring transport activity might hamper the characterisation of the effect of individual molecules and rather portray a cumulative effect of multiple ones.

 

In this project, we suggest to genetically tag candidate genes and express proteins of the plasmodial surface anion channel (PSAC) with various tags to study their trafficking pathways by combined light and electron microscopy methods (CLEM). We will also heterologously express these proteins in a proteoliposome (PL) system rendering the transport molecule(s) in the same orientation as present on the surface of the host erythrocyte (in collaboration with the Maier lab ANU).

 

During the research stay in Berlin, we will generate giant unilamellar vesicles (GUVs) from PLs. In this artificial membrane system, which is free of potentially interfering surface molecules, we can study whether the individual heterologously expressed PSAC components are able to transport known solutes taken up by the NPP via confocal microscopy. The establishment of GUVs from proteoliposomes and solute transport will be performed in the Herrmann lab (HUB).

 

Furthermore, we will study known inhibitors of the NPP (like furosemide and PSAC residual transport inhibitors (PRTs) (2)) and other potential inhibitors from the malaria box to identify whether solute transport can be blocked by the relevant inhibitor (in collaboration with the Saliba lab (ANU).

 

In order to characterise the molecular make-up of the most promising candidates from the inhibitor studies, we will subject proteoliposomes to cryo-electron microscopy studies. Here we aim at resolving the structure of the transport molecule(s) by single particle analysis. Furthermore, we will attempt to visualise the transport molecule(s) in situ by performing cryo-electron tomography on erythrocyte ghosts prepared from P. falciparum-infected cells.

 

These structure-function analyses potentially allow further development of the most effective inhibitor design. Advantages of this indirect targeting approach are that the drug/inhibitor does not have to enter the host cell to reach its target and that the parasite is defenceless against this type of attack (3).

 

 

Keywords topic: New Permeability pathway, Plasmodium falciparum, drug target

 

Keywords methods: protein expression, artificial membrane system, cryo electron microscopy

 

 

(1)   Saliba et al. JBC 1998

(2)   Pain et al, PLOS One, 2016

(3)   Nguitragool et al., Cell, 2011