Molecular characterization of transcriptional promoter sequences of Plasmodium spp

Abstract

Protozoan parasites from the Plasmodium genus have a complex life cycle, with developmental stages in mammalian and arthropod hosts. Stages differentiate by the expression of specific genes, controlled by transcriptional and post-transcriptional mechanisms. Genetic analysis of Plasmodium identified few DNA elements involved in transcriptional control and a few conserved specific Transcription Factors (TFs), suggesting the presence of specific TFs unrelated to previously known DNA binding domains. Plasmodium promoters have been functionally evaluated by transfection of P. falciparum. However, P. falciparum is genetically distant from the other species that infect humans. P. vivax and the other human Malaria parasites P. knowlesi, P. malariae and P. ovale are evolutionary grouped in the primate malaria clade, apart from P. falciparum, a member of the Laverania clade. The evolutionary distance is correlated to biological differences between the species, such as host cell preference, cell invasion mechanisms and gametocyte development. Attempts to characterize P. vivax regulatory sequences using P. falciparum transfection have failed, suggesting differences in transcription control. Recently, P. knowlesi was adapted to continuous in vitro culture, providing a new model for research. In vitro culture of P. knowlesi and P. falciparum allows the design of side-by-side experiments, capable to compare differences and identify conserved features in the species. Preliminary results from our group showed that P. vivax regulatory sequences can be evaluated by P. knowlesi transfection. We analyzed promoter sequences of the P. vivax pvhsp70, pvcrt and pvcam, by transfection of P. knowlesi and P. falciparum with plasmids containing the reporter gene luciferase (luc). All promoter sequences tested were capable to drive luc expression in P. knowlesi. As expected, the pvcam and pvcrt promoter sequences are not recognized by P. falciparum, but the pvhsp70 promoter was capable to induce high expression of luc in both species. The results suggest that pvhsp70 promoter contains elements conserved among three species: P. vivax, P. falciparum and P. knowlesi. To identify functional elements in the pvhsp70 promoter, we will design a series of plasmids with different lengths of the promoter driving luc expression. Plasmids will be used to transfect P. knowlesi and P. falciparum. Plasmids lacking the necessary elements for transcription initiation will produce reduced luc activity. To narrow down the specific sequences responsible for transcriptional control, we will perform gel shift assays - a technique that exploits the fact that DNA bound by protein migrates more slowly through a gel than unbound DNA. We will use extracts from P. falciparum and P. knowlesi to bind to DNA fragments, allowing the identification of any element that interacts with nuclear proteins. To confirm if the sequences have a role in transcription control, we will design new plasmids, deleting these elements or adding these elements to the P. vivax promoters not recognized by P. falciparum (pvcam, pvcrt). Also, we want to identify elements that participate of the stage specific expression control. For this aim, we will use stage synchronized parasites and look for expression changes in the different parasite stages.Finally, in silico analysis will determine if there are multiple promoter region of genes within a single genome that share the functional element, or if there are specific genes, such as hsp70, that have conserved elements across all species. After identification of promoters containing the elements, we will look for associations between the genes. This will help determine why pvhsp70 present heterologous activity. The results combined will provide an insight into the evolution of transcriptional control among Malaria parasites. (AU)