Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival
Posted on: 18 December 2020
Preprint posted on 2 December 2020
Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-21360-8
Categories: cell biology, molecular biology
Background
Many vector-borne pathogens have complex life cycles due to their requirement to transition between insect and mammalian hosts. In these pathogens, cell type differentiation is central to their ability to adapt to different environments. Some parasitic protozoa undergo cell cycle arrest in response to autocrine signals in their host, and can undergo differentiation in response to environmental cues to produce a different cell type that can proliferate. Although in general, little is known about the molecular mechanisms behind these events, the parasites Plasmodium, Trypanosoma brucei and Leishmania have begun to provide some insight into parasite differentiation and survival. Phosphorylation-mediated signal transduction likely plays a pivotal role in Leishmania differentiation, as previous studies have reported protein phosphorylation changes throughout the parasites’ various life cycle stages. Although various studies have explored the role of individual Leishmania protein kinases less than 10% of the kinome has been investigated by genetic and chemical approaches. In the present preprint, Baker et al (1) systemically tagged protein kinases with mNeonGreen fluorescent protein for localization studies, and generated null mutants using CRISPR-Cas9 to study Leishmania survival, differentiation and infection success in vitro and in vivo in both the invertebrate and vertebrate hosts.
Key findings and developments
Generation of gene deletion mutants. The authors began by investigating 204 Leishmania mexicana protein kinases (193 eukaryotic protein kinases and 11 atypical protein kinases). From these, 174 were found to have orthologues in trypanosomes and Leishmania, while 17 were unique to Leishmaniinae (termed LUKs for Leishmaniinae unique kinases). Following identification, the authors attempted to generate gene deletion mutants using CRISPR-Cas9. Gene deletion mutants were successfully produced for 161 protein kinases (that is, 79% of the kinome), while 43 (21%) were found to be essential for promastigotes. 41% of LUKs were essential, which the authors argue is a suggestion that Leishmania promastigotes require these LUKs specifically for life cycle adaptations.
Localization of protein kinases. The authors generated 199 N- or C-terminal mNeonGreen-tagged protein kinases for localization studies in procyclic promastigotes. They used the atlas of Leishmania cellular landmarks and localized proteins to various cell compartments including the cytoplasm, basal body, nucleus, endomembrane, flagellum, lysosome, flagellar pocket, pellicular membrane, cytoplasmic organelles and mitochondrion. Interestingly, the fluorescence signal for some protein kinases varied during the cell cycle.
Phenotypic characterization of gene deletion mutants. The successful mutants were pooled and subjected to Leishmania life cycle progression. Mutants were tested using bar-seq analysis , for their ability to transition through the Leishmania life cycle including promastigotes, metacyclic promastigotes, axenic amastigotes, amastigotes in macrophages and amastigotes in the footpads of mice. The relative growth rate of each mutant was determined by counting the barcodes represented in each time point, and calculating the proportion of each mutant within the population. Outputs for each time point were defined as no loss of fitness, increased relative fitness or decreased relative fitness. Barcodes for each protein kinase were analysed individually, and as clusters, sorting mutants into groups with similar phenotypes taking all time points into account. The authors then used the projection pursuit method to calculate differences between each time point within the series for each of the mutant strains. Various clusters for each of the experimental arms were produced, revealing functional phenotypic groups of protein kinases involved in differentiation from metacyclic promastigote to amastigote, and growth and survival in macrophages and mice. To analyse colonization of the sand fly vector heatmaps were used to show relative loss of fitness, and motility mutants were analysed using transwell migration assays. The latter of which concludes that some of the proteins are fundamental to infection for a reason independent of flagellum defects.
The authors conclude that this unbiased interrogation of protein kinase function in Leishmania allows targeted investigation of organelle-associated signalling pathways required for successful intracellular parasitism.
What I like about this preprint
I have a great interest in parasites, and find the question being targeted in this study, a vital one for our understanding of parasitism. I think the findings in this study, which takes advantage of state-of-the-art molecular tools for phenotypic characterization, will be an interesting baseline for a lot of questions both specific to Leishmania, and general to parasitology.
References
- Baker N and Catta-Preta C, et al, Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival, bioRxiv, 2020.
doi: https://doi.org/10.1242/prelights.26550
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