Development of Genetic Tools in Entamoeba Histolytica

The protozoan parasite Entamoeba histolytica causes dysentery and liver abscesses and is one of the most common causes of death from parasitic disease worldwide. Some parasite genetic determinants involved in pathogenesis have been characterized; however, a complete understanding of the pathogenic mechanisms has not been achieved. A more thorough understanding of the molecular basis of diseases is important as E. histolytica is classified by NIAID as a class B agent of concern for bioterrorism. However, progress has been limited by the paucity of reliable and easy genetic tools. Data from genome projects, microarray experiments and proteomic studies have identified subsets of genes that can be prioritized for genetic analysis. <P>Several methods to genetically manipulate E. histolytica are available but the tools are limited and suffer from time and labor-intensive nature and thus lack of ease for large-scale use, the need for high levels of drug selection and its potential sideeffects, and the lack of methods to disrupt genes on the DNA level due to potential lack of recombination. <P>We aim to develop several novel genetic manipulation tools, which focus on manipulation at the RNA, protein, and DNA levels for application in E. histolytica. First, we will optimize a novel gene silencing using the endogenous RNAi machinery and antisense small RNAs. We will advance this tool by developing this approach to silence (i) using gene fragments, (ii) multiple genes, and (iii) in a regulated manner. <P>Second, we aim to develop a tool to regulate expression on the protein level using a destabilization domain approach (based on the FK506 binding protein and dihydrofolate reductase). We will develop a tool where expression of dominant version of a protein in a regulatable fashion will be a strategy to generate a loss- of function mutant. <P>Third, we will establish a system to integrate foreign DNA into the genome using piggyBac transposase. We aim to generate parasites with random, single integration, so that one gene can be associated to one phenotype.<P> This approach, although high-risk, has succeeded in many different cell types and is worth attempting because integration into the genome will allow novel approaches to be undertaken (gene disruption, promoter trap, etc). In conclusion, these new genetic tools should provide more sophisticated methods of genetic manipulation and will be a great advance for the Entamoeba research community.