Establishment of quantitative RNAi-based forward genetics inEntamoeba histolyticaand identification of genes required for growth

Abstract

Entamoeba histolytica is a globally important pathogen that is dramatically understudied. Its challenging genome has limited tractability. To enable forward genetics, we constructed the first genome-wide E. histolytica RNAi knockdown mutant library. The library is designed to enable deep sequencing analysis for quantitative identification of mutants after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. We performed the first E. histolytica RNAi screen and identified slow growth mutants. Growth phenotypes were reproducible in independently generated mutants. Some of the genes targeted in slow growth mutants had annotated functions consistent with roles in growth or metabolism. Some targeted genes lacked annotation, supporting the power of forward genetics in uncovering gene function. This work opens up the possibility of applying genetics to improve understanding of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms.