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

Abstract

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.

Fig. 1. L. mexicana kinome and conservation in other pathogenic trypanosomatids.

a Representation of eukaryotic (ePK) and phosphatidylinositol 3′ kinase-related kinases (PIKK) protein kinase families, separated by colours defined by primary protein sequence conservation. Image for illustrative purpose only with no phylogenetic significance. Red dots represent protein kinases for which gene deletion mutants could not be generated and are therefore potentially essential genes in procyclic promastigotes. b Phylogenetic tree of trypanosomatids with the number of orthologous protein kinases unique to each node indicated by blue circles and the percentage of potentially essential orthologous protein kinases unique to each node indicated within a red rectangle. c Pie-charts show dispensable (non-essential) and required (potentially essential) protein kinases in procyclic promastigotes, separated into families.

Fig. 2. Generation of the L. mexicana kinome gene deletion library.

a Schematic representation of gene editing by CRISPR–Cas9 in the L. mexicana progenitor cell line showing the integration of repair cassettes containing 30nt homology sites, a unique barcode and antibiotic-resistance genes for puromycin (PAC) and blasticidin (BSD). Diagnostic PCRs for the presence of the coding sequence (CDS) with Oligo 1 and Oligo 2, (depicted in blue), correct integration of the repair cassettes using Oligo 3 and Oligo 4 (depicted in red), and the gene in the endogenous locus with Oligo 5 and Oligo 6 (depicted in green). b Diagnostic PCR for (i) LmxM.25.0853 and (ii) PKAC1 (LmxM34.4010) using primer pairs as above in L. mexicana Cas9T7 (WT) and Populations A and B. c Heat map indicating the presence or absence of target genes after whole-genome sequencing. The ratio of gene coverage to chromosome coverage was used as a measure of gene copy number in selected mutants. A value of 1 represents two alleles of a given gene. d Diagnostic PCR for facilitated gene deletion mutant LmxM.04.0650 using Oligo 1 and 2 for the CDS, Oligo 5 and 6 for the endogenous locus and Oligo 3 and 4 for correct integration. Western blot using anti-myc confirmed the episomal expression of LmxM.04.0650 at the anticipated size of 140 kDa.

Fig. 3. Endogenous tagging of L. mexicana protein kinases.

a Schematic representation of a process for N-terminal tagging. Transfection of the sgRNA (PCR1) directs the double-stranded break immediately upstream of the target CDS and the repair template (PCR2) contains 30 nt at 5′ and 3′ of the fragment for homologous recombination on the target locus to insert the fluorescent tag and an antibiotic-resistance gene. b Bar graph representing the localisation of dispensable and required protein kinases for each organelle. c Pictorial illustration of the 10 localisation groups. An exemplar live-cell image of protein kinases tagged with mNeonGreen for each localisation. Procyclic promastigotes were incubated with Hoechst 33342 for DNA labelling (pink). Scale bar: 5 μm.

Fig. 4. Identification of protein kinases involved in L. mexicana differentiation and infection.

a Schematic illustration of the bar-seq screen design with three experimental arms (EA1, blue; EA2, black; EA3, red) depicting investigation of differentiation to amastigote stages and investigation of protein kinase mutants in sand fly (SF) infection (EA4, green). A pool of gene deletion mutants was generated in procyclic promastigotes (PRO), which were grown in Graces Media at pH 5.5 for 168 h. Cells were then either diluted into amastigote media at 35 °C and grown as axenic amastigotes (AXA) for 5 d or enriched for metacyclic promastigotes. Metacyclic promastigotes (META) were used to either infect macrophages (inMAC) or inoculate mouse footpads (FP). DNA was taken at the indicated time points and the unique barcodes amplified by PCR to apply bar-seq analyses. b Proportion of barcodes across 8 time points, as indicated in a. The trajectories of six exemplar protein kinase mutants have been plotted for the three experimental arms. Values are mean ±  S.D., n = 6 biologically independent samples. Statistical analysis was multiple t-test corrected for multiple comparisons with post-Holm–Sidak method. c Projection pursuit cluster analysis was applied to the trajectories from each experimental arm, grouped into six clusters, each consisting of trajectories with a similar trend. Only clusters resulting from the mouse footpad infection are shown. The image for each cluster shows gene trajectories overlaid with average trends in bold. Trajectories plotted using logged % barcode representation data, normalised to time 0. Heat maps below show % barcode representation data and depict the trend for each individual gene. Gene IDs in red (MCA, MPK1 and MPK2) have documented phenotypes in Leishmania and serve to benchmark the dataset. d The relationship between the clusters is shown on a two-dimensional PCA plot. Colours match the clusters in c.

Fig. 5. Identification of protein kinases involved in colonisation of Lutzomyia longipalpis.

a Heat map of 29 protein kinase mutants that cluster in groups important for the amastigote stage in two of the three experiments using 1 difference clustering. In bold are the genes that have been identified as important for mouse infection in a previous trypanosome study. b Summary showing the protein kinases important for sand fly infection. Control plot (top) shows the relative barcode representation for four control lines with a barcode inserted in the ribosomal locus at three time points post blood meal (PBM) (days 1, 5 and 8). Protein kinases important for L. longipalpis infection plot (bottom) shows the 15 mutants with significant loss of representation by day 8 identified using a multiple t-test, two-stage linear step-up procedure of Benjamin, Krieger and Yekutieli (values are mean ±  S.E., n = 2 biologically independent samples). 7 protein kinases identified as important for both sand fly infectivity and for survival in amastigote stages indicated in bold. c Heat map showing the amastigote stage data for the 8 protein kinase mutants that are important for sand fly infection only. Heat maps show the range of normalised reads. d Plot showing the infectivity of the protein kinase mutants important for the survival of amastigotes only, identified using multiple t-test corrected with post-Holm–Sidak method (values are mean ±  S.E., n = 2 biologically independent samples).

Fig. 6. Identification of protein kinases involved in motility.

a Bar chart showing flagellar length: cell body length ratio for 5 protein kinase-deficient mutants identified with significant loss of representation at the top of the transwell, indicating a motility defect. Error bars indicate standard deviation (n = 50 cells for all samples except Δulk4 where there were 43 cells in the analysis). Mutants with a significant difference in flagella:cell body ratio when compared to Cas9T7 were identified using an unpaired t-test with post-Welch’s correction (***Δulk4 p = 1.07e−25; Δstk36 p = 1.93e−27; Δ29.0600 p = 1.01e−22 and Δ02.0570 p = 1.84e−21). b Graph showing the fraction of the population for each promastigote protein kinase mutant progressively swimming. Five sample chambers were prepared for each cell type. The motile fraction of cells in the control (Cas9T7, n = 2532 cells) and four mutant strains: Δulk4 (n = 2936 cells), Δstk36 (n = 4896 cells), Δ29.0600 (n = 2623 cells) and Δ02.0570 (n = 3532 cells). The error bars represent standard error of the mean/95% confidence intervals and the data points denote results from individual chambers. c Schematic showing the protein structures of the four protein kinase mutants identified in the motility screen. d Localisation of the proteins tagged with mNeonGreen. e Bar-seq trajectories for the four protein kinase mutants for Pool 1, EA3 with loss-of-fitness analysed using multiple t-test corrected with post Holm–Sidak method (values are mean ±  S.D., n = 6 biologically independent samples). f normalised bar-seq reads for these four protein kinase mutants during development in the sand fly (values are mean ±  S.E., n = 2 biologically independent samples).

Fig. 7. Schematic representation of L. mexicana differentiation cycle.

Fifteen protein kinases were identified as required for colonisation of the sand fly. Twenty-nine protein kinases were identified as required for survival as amastigotes in vivo and in vitro.