Unit: Structural Immunology
Director: BENTLEY Graham
Research projects of the unit are centred on the structural and functional study of proteins from pathogens that are of interest for the development of vaccines or for therapeutic strategies that combate infectious diseases. We aim to relate the three-dimensional structure of such antigens and enzymes, determined by X-ray crystallography, to factors important for protective immunity in the case of vaccine candidates, or to understand the molecular function of key enzymes in metabolic pathways that are potential targets for drug design.
Malaria is the most widespread parasite disease, causing more than 2 million deaths each year. With the emergence of resistance to many anti-malarial drugs, the development of a vaccine or new treatments has taken a high priority. Malaria is caused by the unicellular organism, Plasmodium, and all pathologies of the disease are associated with the blood stage of the parasite's life cycle. Plasmodial antigens on the surface of the free parasite, or merozoite, as well as on the surface of the infected red blood cell, are promising vaccine candidates since their can play key roles in biological processes and are accessible to antibodies induced by immune response to the pathogen. In many cases, their function is poorly characterised at the molecular level, and we aim to contribute towards understanding their biological role by relating their 3-dimensional structure to function. In addition, these structural results can be usefully exploited for vaccine development by providing a framework for analysing structural features such as polymorphism and the distribution of important epitopes. Moreover, understanding how these antigens associate with host receptors can aid the design of molecules that block such interactions, thus providing an approach to developing new anti-malarial drugs.
(1) Meroziote Surface Antigens (G.A. Bentley, G. Boulot, L. Larret, J.C. Pizarro, and B. Vulliez-Le Normand, in collaboration with S. Longacre (Immunologie Moléculaire des Parasites, I.P.), F. Nato (Plate-forme: Production de Protéines Recombinantes et d'Anticorps, I.P.), A. Thomas and C. Kochen (Biomedical Primate Reseach Centre, Rijkswijk, Holland))
Surface proteins of the merozoite, the erythrocyte-invading form of Plasmodium, can play an important role in the initial attachment of the parasite to the red blood cell and the subsequent invasion of the latter. Merozoite Surface Protein 1 (MSP1), one of the most widely studied of the Plasmodium surface antigens, is implicated in the erythrocyte invasion process. It is considered to be one of the leading vaccine candidates since it induces immune protection in animal model systems. MSP1 is subjected to several proteolytic cleavages during maturation of the merozoite. During the first phase, it is cleaved to yield four peptide fragments, of which the 42 kDa C-terminal fragment (MSP1-42) remains fixed to the merozoite membrane. During the second phase, occurring at the moment of erythrocyte invasion, MSP1-42 itself is cleaved to yield a polypeptide of molecular weight 11 kDa (MSP1-19). The latter cleavage is essential for erythrocyte invasion, although the mechanism of this process has yet to be elucidated. In order to understand the biological role of MSP1 in the infection of erythrocytes by Plasmodium, we are studying the structure of the recombinant fragments of this protein. Firstly, the structural comparison of MSP1-19 with MSP1-42 could help to explain the importance of the second cleavage step for the penetration of the red blood cell by the merozoite. Secondly, their 3-dimensional structure would give the spatial distribution of polymorphic (dimorphic) residues and the position of protecting epitopes, which is essential information for the optimal conception of candidate vaccine products.
Apical Merozoite Antigen 1 (AMA1) is a type I membrane protein secreted from the apical organelles to the outer surface of the parasite during invasion. Like MSP1, AMA1 is subjected to proteolytic maturation, which appears to be essential for penetration of the red blood cell. AMA1 is also considered a leading vaccine candidate since specific antibodies can inhibit erythrocyte invasion. We have crystallised the ectoplasmic region of the protein and solved its crystal structure.
(2) P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) (C. Badaut, G.A. Bentley, S. Igonet and L. Larret, in collaboration with O. Puijalon (Immunologie Moléculaire des Parasites, I.P.), M. Klinkert (Bernhard Nocht Institute, Hamburg) and D. Arnot (University of Edinburgh)).
After invasion of the erythrocyte, the merozoite of Plasmodium falciparum transforms to the trophozoite and then schizont stages, leading to the liberation of several merozoite progeny upon bursting of the infected host cell. During these two phases, the parasite expresses the protein PfEMP1, which is transported to the surface of the erythrocyte. The protein confers upon the infected erythrocyte the properties of agglutination and of sequestration in diverse receptors present on the surface of endothelial cells of the vasculature. These adhesion phenomena are often associated with severe malaria; PfEMP1 is thus a virulence factor in the disease. The protein is encoded by genes from the var family, for which there are approximately 50 copies per genome (depending on the strain). Different variants of PfEMP1 carry varying numbers and classes of DBL and CIDR domains, which give the molecule (and thus the infected erythrocyte) its specificity for different receptors. We are studying the structural and functional properties of these domains from different variants of the protein.
Structural studies of Tryparedoxin Peroxidase from Trypanosoma cruzi (G.A. Bentley, F. Lema and J.C. Pizarro, in collaboration with A. Cayato, M.D. Pineyro and C. Robello (University of the Republic, Montevideo)
Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in Latin America. To date, there is no vaccine, and currently used drugs have limited effect. In reaction to the oxidative-stress response from the host, T. cruzi detoxifies reactive oxygen species via a metabolic pathway that exploits the thiol trypanothione. Tryparedoxin peroxidase, one of the enzymes of this pathway, catalyses the reduction of reactive oxygen species to water or alcohols by means of redox-active cysteines. We have determined the crystal structure of this enzyme in the reduced state. The active protein exists as a decamer, and shows structural similarity to other peroxiredoxins from other organisms that have been previously described.
Figure 1 : Schematic representation of the homodecamer of Tryparedoxin peroxidase of T. cruzi. Each subunit is shown in a different colour.
Keywords: structural biology, X-ray crystallography, antigenic recognition, antibody structure, Plasmodium antigens