Deletion of a single LeishIF4E-3 allele by the CRISPR-Cas9 system alters cell morphology and infectivity of Leishmania

Rohit Shrivastava, Nitin Tupperwar, Matan Drory-Retwitzer, Michal Shapira

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

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 cytoskeletonand 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.

Original languageEnglish
Article numbere00450-19
JournalmSphere
Volume4
Issue number5
DOIs
StatePublished - 1 Jan 2019

Keywords

  • CRISPR
  • CRISPR-Cas9
  • LeishIF4E-3
  • Leishmania
  • Translation

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Fingerprint

Dive into the research topics of 'Deletion of a single LeishIF4E-3 allele by the CRISPR-Cas9 system alters cell morphology and infectivity of Leishmania'. Together they form a unique fingerprint.

Cite this