The approach of this work is to develop a method for the functional analysis of genes in the malaria parasite and this methodology will then be used to determine the genetic basis of drug resistance in the malaria parasite. This will be a multistep process in which we will first develop a method for he introduction and transient expression of foreign DNA into the parasite, we will then develop a method for the stable introduction of DNA using selectable markers and finally, we will test the role of the malaria mdr-like genes in drug resistance and perhaps in other parasite functions using methods of overexpression and gene knockout by homologous recombination. The malaria parasite presents a unique challenge for transfection in that it is intracellular in most of its life cycle and thus introduced DNA must cross multiple membrane barriers before reaching the parasite nucleus. Because these multiple barriers would be likely to reduce the efficiency of introducing DNA into the parasite we chose to use a parasite stage which was extracellular, this being the female gamete and fertilized zygote. We report the development of a transient transfection vector by constructing a chimeric gene in which the firefly luciferase gene was inserted in frame into the coding region of the pg28 gene of Plasmodium gallinaceum. This plasmid DNA was introduced into P. gallinaceum gametes and fertilized zygotes by electroporation an luciferase expression was assayed after 24 hours. This is the first demonstration of successful introduction and expression of a foreign gene in the malaria parasite and demonstrates the feasibility of this approach to developing methods for the functional analysis of parasite and demonstrates the feasibility of this approach to developing methods for the functional analysis of parasite genes. These results demonstrate transient expression of a reporter gene in the malaria parasite. The method constitutes the first step in the development of a transfection system. Clearly the next step will be the development of stable transfectant using a selectable marker. Further, the pg28 gene is expressed only in the sexual stages and thus either a modification of the flanking sequences to remove stage specific controlling elements or the identification of another gene expressed in other or all life cycle stages will be necessary for analysis of gene expression in asexual or sporozoite stage. Once such transfection vectors are developed, we will test the role of pfmdr genes in drug resistance by a combination of transfection, overexpression and potentially gene knockout experiments. This approach has proven extremely effective in analyzing the role of analogous genes in mammalian systems and more recently in another protozoan parasite, Leishmania sp.
| Emerson, Lyndal R; Nau, Martin E; Martin, Rodger K et al. (2002) Relationship between chloroquine toxicity and iron acquisition in Saccharomyces cerevisiae. Antimicrob Agents Chemother 46:787-96 |
