General Public abstract:
Malaria is a disease that affects millions of people in the world. 1.2 billion persons are at risk, with 198 million cases in 2013 (WHO report 2013-2014). It is prevalent in tropical areas where heat and humidity favors the development of the Anopheles mosquito, the vector which transmits the parasite among humans, or livestock. Insecticide resistant mosquitoes are becoming a problem, as well as drug resistant Plasmodium. The species affecting man are falciparum and vivax, whilst berghei affects rodents. The disease is able to withstand the immune system by evading the attack of antibodies through the use of variable surface molecules. Thus a new wave of parasite is always appearing after a period of clearance. New vaccines are therefore difficult to establish. Our approach has been to try to study the protein Pf-Int which we identified a an member of the recombinase family of enzymes. These molecules can interact with DNA, the information coding support in cells, to change the sequence by recombination, or to peform other tasks to maintain the DNA. We hope therefore to be able to interfere with the ability of the Plasmodium parasite to undergo changes.
Site specific recombinases perform a multitude of DNA maintenance tasks in cells, affecting recombination, repair and replication. The Tyrosine recombinases (Y-SSR) use an active sie tyrosine residue to induce a nucleophilic attack on DNA phosphate backbone leading to a a covalent phospho-tyrosine intermediate, and a free hydroxyl at the 5′ end. This OH can then either reverse the reaction or proceed to strand exchange by attacking the neighboring DNA molecule. The branched intermediate formed is called a Holliday junction. Its isomerization results in either resolution to products or returning to substrate DNA. The target seauences are short palindromic segments that are bound by two copies of recombinase.
A member of the Y-SSR Pf-Int has been identified in the team that possesses all the catalytic residues required to perform the recombination reaction. It has been shown to recognize DNA, and in particular a sequences that belong to the Var family of surface proteins. It also possesses a targeting sequence fo the apicoplast, an organelle derived from the chloroplast of algal origin domesticated by Plasmodium and other apicomplexa. This organelle participates in complex lipid metabolism required by the parasite for cell, tissue, and host transits. The apicoplast also carry a circular 34 Kb genome encoding lipid metabolism genes. Thus Pf-Int could be potentially involved the the management of this circular genome of the apicoplast or in contributing to the generation of variety in the nuclear genome.
Biophysical characterization of Pf-Int by Circulardichroism. DNA binding by Electrophoretic mobility shift. Gene knock out in P. falciparum by double cross over homologous recombination. Stage specific expression of Pf-Int mRNA.