PRJNA672229
Bettadapur et al Entamoeba histolytica RNAi library
While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied relative to other parasites. Given the limitations of existing therapies for amoebiasis and the limitations of our current understanding of the mechanism of disease, new tools are needed. The genetic tractability of E. histolytica has historically been limited, which is largely due to its polyploidy, the large number of gene families, and high A/T content. To enable forward genetics, we constructed and validated the first E. histolytica RNAi knockdown mutant library. This library has genome-wide coverage, with 8307 of 8333 annotated genes represented, and the majority (8301 genes) represented by more than one unique fragment. The 26 genes that are missing are misannotated in the genome. The library design allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We used this library to perform the first RNAi screen in E. histolytica and identified 12 slow growth mutants. Among ORFs targeted in slow growth mutants, many had annotated functions consistent with roles in cellular growth, and/or predicted roles in metabolic pathways. Some targeted ORFs were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. Independently-generated mutants also exhibited slow growth phenotypes, showing that mutant phenotypes were reproducible. Finally, in addition to establishing forward genetics, we uncovered new details of the E. histolytica RNAi pathway. These studies dramatically improve the tractability of E. histolytica and open up the possibility of applying genetics to improve understanding of this important human pathogen.