Role of DNA repair mechanisms in the creation of genomic diversity in Plasmodium falciparum
From the emergence of drug resistance to the seasonal adaptations of influenza, the ability of pathogens to evolve impedes the development of effective, long term interventions for ma ny diseases. In the case of the malaria parasite Plasmodium falciparum , genetic diversity and evolutionary plasticity are major obstacles for disease elimination. Subtelomeric locations harbouring genes coding for variant surface molecules involved in cytoadherence and immune evasion are the most diverse and recombinogenic chromosome regions. Upon drug pressure, apparently any other chromosome region can rapidly develop resistant loci. Much of this variability comes from copy number variations (CNVs), which include gene conversions, deletions, insertions, and duplications/amplifications. CNVs involve changes in the structure of the chromosomes and arise from the repair of double strand breaks (DSBs). P. falciparum faces all the same sources of DSBs as other eukaryotic organisms; therefore DSB repair is expected to be an important aspect of parasite biology particularly in the subtelomeric regions where most of the virulence genes are found. Recent technological breakthroughs have made the investigation of DSBs and their repair pathways in P. falciparum possible. We are developing an endonuclease- based repair assay as a method to directly measure competitive and collaborative actions between distinct pathways and analyze how the repair context and specific attributes of the broken DNA affect this process. We are also performing a genome wide forward genetic approach that can reveal novel factors involved in plasmodial DNA repair and their role in the generation of genome diversity. This knowledge is crucial to estimate the success of future drug and vaccine development.
Functional genomics through transposon-mediated mutagenesis to unravel virulence gene regulation in Plasmodium falciparum
Pathogens have evolved countermeasures to avoid immune clearance and prolong the period of infection in their vertebrate hosts. The type and degree of immune escape strategies depends on the in vivo ‘lifestyle’ the pathogen has adopted. Recent insights using modern molecular biology techniques have shown that this is achieved in protozoans via a coordinated manner of action of different genetic and/or epigenetic factors. The protozoan pathogen Plasmodium falciparum, which infects up to 300 million people causing more than two million deaths each year, undergoes antigenic variation to establish persistent blood stage infection. Several clonally variant gene families undergo antigenic variation in P. falciparum and are expressed during blood stage infection at the surface of infected erythrocytes. One family encoded by 60 var genes expresses the major virulence adhesion surface molecule causing severe malaria (capillary blockages in the brain and other organs mediated by infected erythrocytes). Switching expression between the 60 var gene family members avoids immune clearance of the parasite and prolongs the period of infection, therefore increasing the probability of transmission to the mosquito. Despite major efforts of several laboratories, the molecular mechanism of antigenic variation in P. falciparum remains puzzling due to its complexity. Furthermore, a vast majority of P. falciparum genes have been annotated as coding for hypothetical proteins with no homology to other eukaryotic gene products. This finding suggests that P. falciparum could use unprecedented factors to control monoallelic expression of virulence gene families. We assume that studies in analogy to model systems limit our insight into parasite -specific mechanisms. Thus, we favour an empirical approach combined with a specific genetic screening that can reveal novel mutants affecting var gene regulation.