Background

Figure 1. Erythrocyte invasion.

Erythrocyte invasion: a complex dance between two genomes

All the symptoms and pathology of malaria are initiated when the invasive stage of P. falciparum, called merozoites, infect erythrocytes, leading to the colonisation and eventual destruction of these erythrocytes and the release of a new wave of invasive merozoites 48 hours later. Erythrocyte invasion is critical for parasite survival because P. falciparum is an intracellular parasite – the merozoite can not exist outside of an erythrocyte for any length of time. It is also a complex point of interaction between two genomes and the proteins that they encode.

Erythrocyte invasion consists of several phases that depend on erythrocyte and merozoite proteins. Invasion begins with a reversible recognition phase, which is followed by the formation of an irreversible junction between the merozoites and the erythrocyte surfaces. This is known as the apical junction because it involves the tip or apex of the merozoite. Both of these steps involve interactions between proteins on the erythroycte and merozoite cell membranes, which are generally referred to as receptors and ligands respectively. Work by many labs has identified multiple receptors and ligands that may be involved in recognition and attachment, but in very few cases is it known which receptor binds to which ligand.

After the apical junction is formed, it splits and is driven backwards along the length of the merozoite, which rapidly leads to the complete internalisation of the merozoite within the erythrocyte. This entry step is driven entirely by a protein scaffolding system within the merozoite called an actin-myosin motor, which is in turn anchored to the cellular protein skeleton called the cytoskeleton, by three membrane proteins named PfMTIP, PfGAP45 and PfGAP50. The cell modifies the structure of these proteins after they are made and hence alters their activity, and these proteins may be key players in the regulation of erythrocyte invasion itself.

Variation in invasion pathways

Erythrocyte invasion is a relatively flexible phenomenon and many of the receptor-ligand interactions involved in attachment and recognition are to some extent interchangeable. Natural genetic variation in both the parasite and host adds an additional layer of complexity to these interactions. Genetic variation of this nature can have relatively major effects, for example dictating which primate erythrocytes that P. falciparum merozoites can recognise, and therefore impacting on parasite-host species specificity. Genetic variation in receptors and ligands may also have much more subtle effects on invasion, perhaps influencing growth rates and pathogenicity in specific individuals.

Research

Our aims

The Rayner team aims to develop a better understanding of which receptors bind to which ligands during the recognition and attachment phases of invasion. We also work in concert with the unique genotyping and sequencing approaches available at the Sanger Institute to understand how how natural genetic variation in both the host and the parasite affect both receptor-ligand binding and the overall efficiency of erythrocyte invasion. In this way we aim to link specific observable characteristics (or phenotypes) with the DNA sequence (or genotypes) of malaria parasites and their hosts. Finally we also aim to understand how subtle changes in specific proteins, such as the membrane proteins that anchor the actin-myosin motor, may regulate erythrocyte invasion. These subtle modifications and the enzymes that drive them are potential anti-malarial drug targets.

Figure 2. Haemagglutinin tagged PfGAP45 (red) co-localizes with endogenous PfGAP45 (green) at the periphery of each merozoite (the nucleus of each merozoite is shown by a blue DNA stain).

Our approach

To achieve our aims we use a variety of approaches, many of which make use of the unique strengths of the Sanger Institute. Tiny changes in the DNA sequence called single nucleotide polymorphisms (SNPs) are known to be responsible for a lot of the variation seen in the DNA sequences of organisms. Genome-wide studies in the Sanger Institute's malaria programme are now identifying numerous SNPs within both human erythrocyte receptors and P. falciparum invasion ligands (Malaria Programme: Kwiatkowski group), but their effect on receptor-ligand binding or invasion is not yet known. We are using new high-througput phenotyping approaches to assess the effect that these SNPs have on specific steps during erythrocyte invasion.

We are also using experimental genetic approaches to dissect the molecular roles of specific P. falciparum proteins in the process of erythrocyte invasion. In these experiments we artifically label and alter the invasion proteins in order to follow them by microscopy during P. falciparum development and invasion. We are also able to use large-scale protein-based (proteomic) techniques to undersand how the parasite modifies the proteins to regulate the invasion process. An example of this approach is shown below in Figure 2. Here we have fluorescently labelled several P. falciparum membrane proteins in two colours, and the cell nucleus in blue, to show how the proteins interact in growing and dividing merozoites.

Selected Publications

• Effects of calcium signaling on Plasmodium falciparum erythrocyte invasion and post-translational modification of gliding-associated protein 45 (PfGAP45).

  Jones ML, Cottingham C and Rayner JC

  Molecular and biochemical parasitology 2009;168;1;55-62

  PUBMED: 19576251; DOI: 10.1016/j.molbiopara.2009.06.007; PMC: 2754074

• Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex.

  Jones ML, Kitson EL and Rayner JC

  Molecular and biochemical parasitology 2006;147;1;74-84

  PUBMED: 16513191; DOI: 10.1016/j.molbiopara.2006.01.009

• Evolution of human-chimpanzee differences in malaria susceptibility: relationship to human genetic loss of N-glycolylneuraminic acid.

  Martin MJ, Rayner JC, Gagneux P, Barnwell JW and Varki A

  Proceedings of the National Academy of Sciences of the United States of America 2005;102;36;12819-24

  PUBMED: 16126901; DOI: 10.1073/pnas.0503819102; PMC: 1200275

• Dramatic difference in diversity between Plasmodium falciparum and Plasmodium vivax reticulocyte binding-like genes.

  Rayner JC, Tran TM, Corredor V, Huber CS, Barnwell JW and Galinski MR

  The American journal of tropical medicine and hygiene 2005;72;6;666-74

  PUBMED: 15964948

• A Plasmodium falciparum homologue of Plasmodium vivax reticulocyte binding protein (PvRBP1) defines a trypsin-resistant erythrocyte invasion pathway.

  Rayner JC, Vargas-Serrato E, Huber CS, Galinski MR and Barnwell JW

  The Journal of experimental medicine 2001;194;11;1571-81

  PUBMED: 11733572; PMC: 2193530

• Two Plasmodium falciparum genes express merozoite proteins that are related to Plasmodium vivax and Plasmodium yoelii adhesive proteins involved in host cell selection and invasion.

  Rayner JC, Galinski MR, Ingravallo P and Barnwell JW

  Proceedings of the National Academy of Sciences of the United States of America 2000;97;17;9648-53

  PUBMED: 10920203; DOI: 10.1073/pnas.160469097; PMC: 16919