Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth
| العنوان: | Establishment of quantitative RNAi-based forward genetics in Entamoeba histolytica and identification of genes required for growth |
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| المؤلفون: | Tammie S. Y. Tam, Hannah W. Miller, Rene L. Suleiman, Samuel S. Hunter, Wesley Huang, Maura C. Ruyechan, Akhila Bettadapur, Matthew L. Settles, Charles G. Barbieri, Katherine S. Ralston |
| المساهمون: | Singh, Upinder |
| المصدر: | PLoS Pathogens, Vol 17, Iss 11, p e1010088 (2021) PLoS Pathogens PLoS pathogens, vol 17, iss 11 |
| بيانات النشر: | Public Library of Science (PLoS), 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | Mutant, Protozoan Proteins, Genome, Biochemistry, RNA interference, Amoebas, Cloning, Molecular, Biology (General), Protozoans, Staining, biology, Entamoebiasis, Entamoeba histolytica, Eukaryota, High-Throughput Nucleotide Sequencing, Genomics, Specimen preparation and treatment, Nucleic acids, Infectious Diseases, Genetic interference, Medical Microbiology, Gene Knockdown Techniques, Protozoan, RNA Interference, Epigenetics, Biotechnology, Research Article, QH301-705.5, 1.1 Normal biological development and functioning, Immunology, Computational biology, DNA construction, Transfection, Research and Analysis Methods, Microbiology, Deep sequencing, Vaccine Related, Entamoeba Histolytica, Underpinning research, Biodefense, Virology, Genetics, Animals, Molecular Biology Techniques, Gene, Molecular Biology, Gene Library, Prevention, Organisms, DAPI staining, Molecular, Biology and Life Sciences, DNA, DNA, Protozoan, RC581-607, biology.organism_classification, Forward genetics, Parasitic Protozoans, Emerging Infectious Diseases, Plasmid Construction, Nuclear staining, Mutation, RNA, Parasitology, Generic health relevance, Gene expression, Immunologic diseases. Allergy, Genome, Protozoan, Cloning, Genome-Wide Association Study |
| الوصف: | 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. Author summary Entamoeba histolytica is a globally important pathogen that is dramatically understudied. One of the major limitations of this organism is its challenging genome. RNAi is the state-of-the-art tool for genetic manipulation in E. histolytica, though the RNAi pathway has several noncanonical features. Here, we harnessed the RNAi pathway to enable RNAi-based forward genetics for the first time in this organism. We validated the RNAi library by performing the first E. histolytica RNAi screen and identified slow growth mutants. We showed that independently-generated mutants also exhibited slow growth phenotypes, and we characterized protein localization and domains for some of the identified slow growth genes. The RNAi library that we constructed enables modern, quantitative Illumina deep sequencing analysis to identify mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify full-length gene fragments inferred from read mapping. Our approach differs from previous approaches for analysis of RNAi screens, and it better represents the actual DNA fragments and their quantities. This study dramatically improves the tractability of this important pathogen. Moreover, the strategies behind this RNAi library, and its analysis, are novel, and can be applied to other organisms. |
| وصف الملف: | application/pdf |
| اللغة: | English |
| تدمد: | 1553-7374 1553-7366 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::6adc2262c4f898a71f6cb9ffae17ae6f https://doaj.org/article/35506d785cd04428b109dd5265320e89 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....6adc2262c4f898a71f6cb9ffae17ae6f |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15537374 15537366 |
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