Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth

Abstract

While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E. histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E. histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E. histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual 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 pathogen.

Growth and Genetic Manipulation of Entamoeba histolytica

“…no. GA1200) pTrigger plasmid (see Bettadapur et al, 2021;Morf et al, 2013) Forward and reverse primers with homology to insert and backbone (for Gibson assembly) DH5α competent Escherichia coli (e.g., New England Biolabs, cat. no.…”

“…Although the original description of the pTrigger plasmid described using GOI fragments that were both in the forward orientation and in frame, we have empirically determined that the insert may be either in or out of frame, and it may be in either the forward or reverse orientation for silencing to be effective (Bettadapur et al, 2021).…”

“…An advantage of this strategy for E. histolytica transfection is that it uses less plasmid DNA and fewer trophozoites than available electroporation protocols. The stable transfection efficiency of E. histolytica using this method is approximately one in a thousand cells (Bettadapur et al, 2021), though this is expected to vary depending on the plasmid construct.…”

“…pTrigger contains 132 bp of the trigger gene EHI_048600 directly before a SmaI restriction enzyme cut site added to make cloning inserts easier (Fig. 4; Bettadapur et al, 2021). It may be necessary to check your specific E. histolytica culture for endogenous knockdown of this particular trigger gene.…”

“…a. Roughly 500 to 1000 bp in length (Bettadapur et al, 2021;Khalil et al, 2016;Morf et al, 2013); b. Low homology to other genes to minimize off-target silencing; c. Smaller than the size of the GOI if possible (which helps at a later step by allowing for the design of primers that sit outside of the GOI fragment for RT-PCR analysis of gene knockdown); d. Whether the GOI fragment will be cloned into pTrigger in the forward or reverse orientation (Bettadapur et al, 2021;Morf et al, 2013); and e. Whether the GOI fragment will be cloned into pTrigger in or out of frame (Bettadapur et al, 2021;Morf et al, 2013).…”