10 July 2014
By Zenon Zenonos
An erythrocyte being invaded by a merozoite. Credit: ©1979 Rockefeller University Press. Journal of Experimental Medicine. 149:172-184. doi:10.1084/jem.149.1.172
All the evidence seems to suggest that the proteins found on the surface of the malaria parasite when it invades red blood cells are important vaccine candidates. However, the unique structure of these proteins makes them difficult to reproduce and study in the lab, and hence we haven’t been able to uncover the exact function for most of them.
As a result, and despite the high rates of morbidity and mortality due to malaria infection, no malaria vaccine has been licensed to date. Until we know what these proteins are doing, we won’t be able to choose which ones to target to make new drugs and vaccines.
The malaria parasite Plasmodium falciparum is the most virulent among the six Plasmodium species that are able to infect humans, causing over a million deaths annually, especially in children under the age of five. It’s transmitted to humans by the bite of a female Anopheline mosquito. Once in the body, the parasite infects the liver cells, within which it replicates and differentiates to form thousands of invasive merozoites - the blood stage form of the parasite. After about 10 days in the liver, merozoites are released into the blood stream where they infect circulating red blood cells.
Red blood cell invasion by merozoite is an obligatory step in the parasite’s life cycle. The proteins exposed on merozoite’s cell surface are believed to play pivotal role in the invasion process and because they are directly accessible to human immune system, they have been long-standing targets for therapeutic interventions and vaccine development.
However, the precise function of most of the proteins displayed on merozoite cell surface is largely unclear. Progress in understanding their exact function has been hindered by technical challenges related with expressing these proteins in a biochemically active and functional form.
Plasmodium falciparum proteins are very unusual in comparison to proteins of other organisms; they have very repetitive amino acid stretches and are encoded by very unusual DNA sequences. This makes them notoriously difficult to produce and study outside of their normal environment.
To address these problems, a method of expressing artificial versions of the merozoite cell-surface and secreted proteins, was previously developed by our lab. The method utilises a mammalian expression system – instead of bacteria, which is the system most commonly used to produce proteins in the laboratory – to increase the chances of getting correctly folded, functional proteins. By taking this approach, a library of 42 merozoite cell surface and secreted proteins was successfully created in previous studies in our lab.
However, it has been clear that the merozoite cell surface includes many more proteins that were not included in our initial library. To expand our previously constructed protein library, we used genome-wide expression profiling and identified 20 additional merozoite cell surface and secreted protein candidates that we artificially produced at high quantities, by using our previous methodology.
In order to analyse and compare the 20 proteins we had found, we purified all 20 proteins in parallel by using a high-throughput, custom built, piston-driven purification platform. It’s important to purify all proteins at once to avoid variation between experiments. Moreover, we demonstrated that most of the 20 purified proteins were recognised by antibodies obtained from malaria patients, suggesting that they are biochemically active; this means that further studies into their functions may now be conducted.
This library of 62 proteins brings us closer to our eventual aim of compiling a set of proteins that is representative of the merozoite surface. All plasmid constructs, which allow researchers to create artificial versions of proteins for laboratory analysis, are freely available to the global research community through Addgene a non-profit, open access plasmid repository. We believe that these plasmids will be a valuable resource for basic research and will aid the efforts to develop an effective malaria vaccine.
The work described above was done when Zenon Zenonos was a PhD student in the Cell Surface Signalling laboratory where he worked under the supervision of Gavin Wright on identifying novel receptor-ligand pairs involved in erythrocyte invasion by P. falciparum. Currently, Zenon is a postdoctoral fellow in The Sanger Institute Malaria Programme, under Julian Rayner, and he works on P. falciparum molecular genetics.
- Zenonos, Z et al (2014). Towards a comprehensive Plasmodium falciparum merozoite cell surface and secreted recombinant protein library. Malaria Journal. doi:10.1186/1475-2875-13-93