Differentiation between distinct stages is fundamental for the life cycle of intracellular protozoan parasites and for transmission between hosts, requiring stringent spatial and temporal regulation. Here, we apply kinome-wide gene deletion and gene tagging in Leishmania mexicana promastigotes to define protein kinases with life cycle transition roles. Whilst 162 are dispensable, 44 protein kinase genes are refractory to deletion in promastigotes and are likely core genes required for parasite replication. Phenotyping of pooled gene deletion mutants using bar-seq and projection pursuit clustering reveal functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, growth and survival in macrophages and mice, colonisation of the sand fly and motility. This unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.
- MeSH
- biologické modely MeSH
- buněčná diferenciace * MeSH
- CRISPR-Cas systémy genetika MeSH
- delece genu MeSH
- flagella enzymologie MeSH
- Leishmania mexicana cytologie enzymologie MeSH
- leishmanióza parazitologie patologie MeSH
- mutace genetika MeSH
- myši inbrední BALB C MeSH
- protein Cas9 metabolismus MeSH
- proteinkinasy genetika metabolismus MeSH
- proteom metabolismus MeSH
- Psychodidae parazitologie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In our previous work we established a T7 polymerase-driven Tetracycline-inducible protein expression system in Leishmania mexicana (Biagi, 1953). We used this system to analyse gene expression profiles during development of L. mexicana in procyclic and metacyclic promastigotes and amastigotes. The transcription of the gene of interest and the T7 polymerase genes was significantly reduced upon cell differentiation. This regulation is not locus-specific. It depends on untranslated regions flanking open reading frames of the genes analysed. In this paper, we report that the previously established conventional inducible protein expression system may not be suitable for studies on differentiation of species of Leishmania Ross, 1903 and protein expression systems might have certain limitations.
