Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>

ABSTRACT The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical featur...

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Autores principales:	Rohit Shrivastava, Nitin Tupperwar, Matan Drory-Retwitzer, Michal Shapira
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	CRISPR CRISPR-Cas9 LeishIF4E-3 Leishmania translation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a50ded8260b44a42bf9e55e6aaeea474
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spelling	oai:doaj.org-article:a50ded8260b44a42bf9e55e6aaeea4742021-11-15T15:27:32ZDeletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>10.1128/mSphere.00450-192379-5042https://doaj.org/article/a50ded8260b44a42bf9e55e6aaeea4742019-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00450-19https://doaj.org/toc/2379-5042ABSTRACT The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in Leishmania mexicana promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in de novo protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/−) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence. IMPORTANCE Leishmania species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. Leishmania parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for Leishmania, we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.Rohit ShrivastavaNitin TupperwarMatan Drory-RetwitzerMichal ShapiraAmerican Society for MicrobiologyarticleCRISPRCRISPR-Cas9LeishIF4E-3LeishmaniatranslationMicrobiologyQR1-502ENmSphere, Vol 4, Iss 5 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR CRISPR-Cas9 LeishIF4E-3 Leishmania translation Microbiology QR1-502
spellingShingle	CRISPR CRISPR-Cas9 LeishIF4E-3 Leishmania translation Microbiology QR1-502 Rohit Shrivastava Nitin Tupperwar Matan Drory-Retwitzer Michal Shapira Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
description	ABSTRACT The genomes of Leishmania and trypanosomes encode six paralogs of the eIF4E cap-binding protein, known in other eukaryotes to anchor the translation initiation complex. In line with the heteroxenous nature of these parasites, the different LeishIF4E paralogs vary in their biophysical features and their biological behavior. We therefore hypothesize that each has a specialized function, not limited to protein synthesis. Of the six paralogs, LeishIF4E-3 has a weak cap-binding activity. It participates in the assembly of granules that store inactive transcripts and ribosomal proteins during nutritional stress that is experienced in the sand fly. We investigated the role of LeishIF4E-3 in Leishmania mexicana promastigotes using the CRISPR-Cas9 system. We deleted one of the two LeishIF4E-3 alleles, generating a heterologous deletion mutant with reduced LeishIF4E-3 expression. The mutant showed a decline in de novo protein synthesis and growth kinetics, altered morphology, and impaired infectivity. The mutant cells were rounded and failed to transform into the nectomonad-like form, in response to purine starvation. Furthermore, the infectivity of macrophage cells by the LeishIF4E-3(+/−) mutant was severely reduced. These phenotypic features were not observed in the addback cells, in which expression of LeishIF4E-3 was restored. The observed phenotypic changes correlated with the profile of transcripts associated with LeishIF4E-3. These were enriched for cytoskeleton- and flagellum-encoding genes, along with genes for RNA binding proteins. Our data illustrate the importance of LeishIF4E-3 in translation and in the parasite virulence. IMPORTANCE Leishmania species are the causative agents of a spectrum of diseases. Available drug treatment is toxic and expensive, with drug resistance a growing concern. Leishmania parasites migrate between transmitting sand flies and mammalian hosts, experiencing unfavorable extreme conditions. The parasites therefore developed unique mechanisms for promoting a stage-specific program for gene expression, with translation playing a central role. There are six paralogs of the cap-binding protein eIF4E, which vary in their function, expression profiles, and assemblages. Using the CRISPR-Cas9 system for Leishmania, we deleted one of the two LeishIF4E-3 alleles. Expression of LeishIF4E-3 in the deletion mutant was low, leading to reduction in global translation and growth of the mutant cells. Cell morphology also changed, affecting flagellum growth, cell shape, and infectivity. The importance of this study is in highlighting that LeishIF4E-3 is essential for completion of the parasite life cycle. Our study gives new insight into how parasite virulence is determined.
format	article
author	Rohit Shrivastava Nitin Tupperwar Matan Drory-Retwitzer Michal Shapira
author_facet	Rohit Shrivastava Nitin Tupperwar Matan Drory-Retwitzer Michal Shapira
author_sort	Rohit Shrivastava
title	Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
title_short	Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
title_full	Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
title_fullStr	Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
title_full_unstemmed	Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>
title_sort	deletion of a single leishif4e-3 allele by the crispr-cas9 system alters cell morphology and infectivity of <italic toggle="yes">leishmania</italic>
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/a50ded8260b44a42bf9e55e6aaeea474
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Deletion of a Single LeishIF4E-3 Allele by the CRISPR-Cas9 System Alters Cell Morphology and Infectivity of <italic toggle="yes">Leishmania</italic>

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