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Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival

Baker N., Catta-Preta C.M.C., Neish R., Sadlova J., Powell B., Alves-Ferreira E.V.C., Geoghegan V., Carnielli J.B.T., Newling K., Hughes C., Vojtkova B., Anand J., Mihut A., Walrad P.B., Wilson L.G., Pitchford J.W., Volf P., Mottram J.C.

Preprint posted on December 02, 2020 https://www.biorxiv.org/content/10.1101/2020.09.06.279091v2

Article now published in Nature Communications at http://dx.doi.org/10.1038/s41467-021-21360-8

Insights into Leishmania differentiation and survival

Selected by Mariana De Niz

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.

Figure 1. L. mexicana kinome and conservation in other pathogenic trypanosomatids. Pie- charts show dispensable and required (potentially essential) protein kinases in procyclic promastigotes, separated into families. (Ref. 1).

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

  1. Baker N and Catta-Preta C, et al, Systematic functional analysis of Leishmania protein kinases identifies regulators of differentiation or survival, bioRxiv, 2020.

 

Posted on: 18th December 2020

doi: https://doi.org/10.1242/prelights.26550

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Author's response

Nicola Baker, Carolina Catta-Preta and Jeremy Mottram shared

Open questions 

1.Barcoding has been used for the phenotypic characterization of other parasites, such as Plasmodium berghei, in its multiple stages and hosts. An important question is whether and how each mutant affects one another when present in pools, as opposed to independent infections? Of course the advantage of pool infections is the high-throughput and that it respects the 3R principles of animal use. However, can competition, inhibition, and other forms of influence between parasite populations be excluded?

Within any competition assay involving parasite populations there may be dominant negative or dominant positive effects on individual mutants within the pool.  Pooled library screens are most suited to identify genes involved in parasite-intrinsic functions, whereas functions extrinsic to the parasite might be compensated for by other mutants in the pool.  For example, the phenotype of a mutant lacking a protein kinase that would normally be released in extracellular vesicles to influence the host immune response might be masked by other mutants in the population releasing the protein kinase.  One way to control for this would be to randomly reassort pools of mutants and repeat the phenotype analysis.

2.Your observations on the changes of signal localization depending on the cell cycle are very interesting. Are these changes conserved in other organisms? What is the basis for such changes?

Changes in protein localisation have been reported in many organisms, from humans to protozoa.   The human cyclin‐dependent kinase 5 (CDK5) for instance is localized to the cytoplasm, but once its non-cyclin regulatory subunits p35 and p39 are myristoylated the protein is attached to the plasma membrane and perinuclear membranes (Asada et al., 2008 – PMID: 18507738), while phosphorylation of p39 reduces nuclear localisation (Asada et al., 2012 – PMID: 22467861). In Trypanosoma brucei, a parasite phylogenetically related to Leishmania, kinetochore proteins are dynamically phosphorylated during the cell cycle, and KKT1 for example can be found in the cytoplasm in early-cell cycle phases or the kinetochore from S-phase through G2/M, when it is also highly-phosphorylated (Benz and Urbaniak, 2019 – PMID:31830130). Further investigations are necessary to understand the dynamics of cell cycle dependent localisation in Leishmania, and identify post-translational modifications and regulators involved in this mechanism.

3.Another broad question: for the in vivo characterization, you used Balb/c mice. Are there significant differences in infection outcomes in Balb/c as opposed to other mouse strains (inbred or outbred), that could explain some involvement of protein kinases with their interaction with the host’s immune system?

The kinetics of development of cutaneous lesions can differ in strains of mice, reflecting the differences in innate immune response.  It would certainly be interesting to carry out infection of the pooled protein kinase gene deletion library in different mouse strains and transgenic animals deficient in immune function.  This will give insights into host-parasite interaction.

4.Regarding motility, one of your study’s conclusions is that some protein kinases are fundamental to infection for reasons independent to flagellum defects. Can you expand further on the outcomes of infection with respect to flagellum defects- namely to what extent is motility important, and how is each time point you studied, affected by motility defects?

A study by Beneke et al (PMID: 31242261) found mutants with short flagella to be less fit at colonising the sand fly gut, leading to a hypothesis that motility is required for successful infection. In our study we used a transwell assay to identify four protein kinase mutants that had reduced motility and a short flagellum. Of these, only two showed reduced fitness in the sand fly colonisation assay. We hypothesised that LmxM.29.0600 and LmxM.02.0570 null mutants retained some motility despite their short flagella, however, we found this not to be the case.  Colonisation of the sand fly was studied by taking genomic DNA from the whole fly at three timepoints reflecting the presence of the mutant in the bloodmeal (day 1), in the sandfly midgut after defecation (day 5) and at the peak of metacyclogenesis (day 8). A significant loss of representation at day 5 and 8 suggests that a mutant could not colonise the sand fly after defecation. The presence of LmxM.29.0600 and LmxM.02.0570 null mutants within the pooled population at days 5 and 8 indicates that they were able to colonise the sandfly midgut despite no motility. We plan to infect sand flies with the individual mutants to see if they undergo normal metacyclogenesis and can colonise the foregut, as this will be necessary to help understand the impact of motility on colonisation.

5.What are the main differences and similarities you found between your findings in Leishmania with respect to Trypanosoma brucei, and/or to the Apicomplexans regarding parasite requirements for differentiation and survival?

The life cell cycle of protozoa reflects their adaptations to parasitism, including strategies of immune evasion, intracellular or extracellular life. Whilst some protein kinases can clearly be defined as having orthologues between species, others are unique.  The CRK (Cdc-2 related kinase) family are cyclin-dependent kinases known for their role in cell cycle regulation, mRNA processing and differentiation, and are represented by 11 proteins in Trypanosoma and Leishmania, from which 6 are essential to infective bloodstream forms of T. brucei (CRK1-3;6,9,12), (Jones et al., 2014 – PMID: 24453978).  In Leishmania promastigotes CRK1-3;9,11,12 are most likely essential, implying similar roles across trypanosomatids. The kinomes of Toxoplasma gondii and Plasmodium are poorly conserved with trypanosomatids, making it particularly difficult to compare functions.

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