mosquito invasion of malaria research…
When we named our first liquid handler ‘mosquito’, we never expected that one of our mosquito instruments would be used to determine the structure of a malaria parasite protein!
Malaria is a mosquito-borne infectious disease caused by parasites of the Plasmodium type. UNICEF states that malaria kills one child every 30 seconds!
The multiple stages of a clever parasite
Commonly, the disease is transmitted by the bite of an infected female Anopheles mosquito. This bite introduces the parasites from the mosquito’s saliva into a person’s blood. However what is not commonly known is that the parasites then travel to the liver where they mature and reproduce before entering the blood stream again and invading red blood cells. The parasite multiplies in the red blood cells which then burst open causing the first symptoms of fever (Figure 1). Most deaths are caused by Plasmodium falciparum due to the cerebral effects from this parasite. Although P. vivax, P. ovale, and P. malariae generally cause a milder form of malaria, these clever parasites can remain dormant in the liver for many years causing disease later in life.
Figure 1: Life cycle of the malaria parasite (courtesy of L.A. Dept of Public Health http://publichealth.lacounty.gov/acd/VectorMalaria.htm)
How crystallography can help vaccine development
Vaccine development against malaria has been very challenging due to the multiple stages of parasite growth as described.
Obviously, invasion of host red blood cells is an essential stage to the life cycle of P. falciparum parasites therefore exploiting this interaction has been a hot topic for vaccine research. Multiple proteins are responsible for the binding of the parasite to the red blood cells, one of which is the reticulocyte-binding protein homologue (RH). Dr. Wright and her colleagues have recently published in Nature (17 August 2014) the first structural insight into red blood cell binding to RH5 from Plasmodium falciparum (PfRH5). Excitement about PfRH5 as a next-gen blood stage malaria vaccine target has been generated due to a number of in vitro studies. Antibodies against one PfRH5 variant neutralised parasites of all heterologous strains tested and anti-PfRH5 monoclonal antibodies that prevent parasite growth in vitro can block the PfRH5-basigin (a protein found in red blood cells) interaction.
Structural studies involved a number of difficult stages. Initially a modified PfRH5 protein was mixed with basigin or fragments of growth-inhibitory monoclonal antibodies to form complexes that were trimmed and methylated before crystallisation. Crystallisation was achieved using vapour diffusion in sitting drops employing a mosquito LCP robot (TTP Labtech) to pipette 100 nL drops of each protein and 100 nL of solutions from commercial crystal screens. Crystal seeds were generated from the screen at two temperatures. 50 nL of new crystal seeds were then added to 100 nL of original protein complex along with 50 nL of well solution and 50 nL Silver Bullets. Final crystals were obtained at Diamond Light Source and analysed for structural identity.
PfRh5 adopts a rigid, flat, ‘kite-shaped’ architecture with a twofold rotation symmetry and no similarity to known structures. Sequence homology identifies this fold at the N terminus of other RH proteins, where it is likely to act as a ligand-binding module.
Immunogens containing these regions of PfRH5 could be important components of a vaccine to prevent P. falciparum erythrocyte invasion and thereby lead to the prevention of malaria.
Dr. K.E. Wright works in Dr. Matt Higgins group at the Department of Chemistry, University of Oxford, UK. The group studies interactions between proteins from parasites that cause disease and molecules from their human host. They use structural methods to understand how these proteins interact with specific ligands on human tissues, and also attempt to develop agents that block these interactions.