Culture and transfection of an emerging malaria pathogen

02 January 2013

Researchers at NIMR have adapted Plasmodium knowlesi to continuous culture in human erythrocytes, providing a new, genetically tractable model for malaria research. The work is published in the Proceedings of the National Academy of Sciences, USA.

Malaria kills over 1 million people each year and is an important cause of poverty. The development in 1976 of a system of continuous culture for Plasmodium falciparum, the causative agent of the most widespread form of severe malaria, was a milestone for research into this parasite. It enabled genetic modification of the parasite and high-throughput drug screening, and has led to fundamental advances in our understanding of malaria parasite biology. Adaptation of the other four human malaria parasite species to in vitro culture has proved to be more challenging, and until now it has not been possible for any of them to be maintained in continuous culture.

The simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of SE Asia and is closely related to P. vivax, which is the major cause of malaria outside Africa. Much of the early work on the mechanics of red blood cell invasion by the malaria parasite used P. knowlesi. Its short (~24 hour) life cycle, the relatively large size of its merozoites and its amenability to genetic manipulation make it a valuable tool for studying the cell biology of the parasite. However, in the absence of an in vitro culture system, research on P. knowlesi has been restricted to just a few laboratories world-wide with access to primate facilities.

Rob Moon, who holds a joint MRC Centenary Award Fellowship in the labs of Tony Holder and Mike Blackman (pictured) in NIMR’s Division of Parasitology, collaborated with researchers at KAUST in Saudi Arabia. Together they have adapted a P. knowlesi line to continuous culture in human red blood cells, and have demonstrated that the culture-adapted clones are highly amenable to genetic manipulation.

We have achieved the highest reported transfection efficiency for any malaria parasite. This will for the first time allow analysis of transgenic parasites within the first generation following transfection, and provides the first possibility of a genome-wide knock-out project in a human malaria pathogen.

Rob Moon

Importantly, the human-adapted P. knowlesi clones are still capable of invading and replicating within monkey erythrocytes, opening up the future potential for both in vivo and in vitro experiments. This has not previously been possible for any human malaria pathogen.

Mike Blackman

Click image to view at full-size

Adaptation of the P. knowlesi A1 strain to continuous culture in human erythrocytes. Strategy used to adapt the parasites to growth exclusively in human RBC.

Original article

Robert W Moon, Joanna Hall, Farania Rangkuti, Yung Schwen Ho, Neil Almond, Graham H Mitchell, Arnab Pain, Anthony A Holder & Michael J Blackman (2012)

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Proceedings of the National Academy of Sciences, USA. Epub ahead of print.  Publisher abstract

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