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An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria

Abstract

The most severe form of human malaria is caused by Plasmodium falciparum. Its virulence is closely linked to the increase in rigidity of infected erythrocytes and their adhesion to endothelial receptors, obstructing blood flow to vital organs. Unlike other human-infecting Plasmodium species, P. falciparum exports a family of 18 FIKK serine/threonine kinases into the host cell, suggesting that phosphorylation may modulate erythrocyte modifications. We reveal substantial species-specific phosphorylation of erythrocyte proteins by P. falciparum but not by Plasmodium knowlesi, which does not export FIKK kinases. By conditionally deleting all FIKK kinases combined with large-scale quantitative phosphoproteomics we identified unique phosphorylation fingerprints for each kinase, including phosphosites on parasite virulence factors and host erythrocyte proteins. Despite their non-overlapping target sites, a network analysis revealed that some FIKKs may act in the same pathways. Only the deletion of the non-exported kinase FIKK8 resulted in reduced parasite growth, suggesting the exported FIKKs may instead support functions important for survival in the host. We show that one kinase, FIKK4.1, mediates both rigidification of the erythrocyte cytoskeleton and trafficking of the adhesin and key virulence factor PfEMP1 to the host cell surface. This establishes the FIKK family as important drivers of parasite evolution and malaria pathology.

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Fig. 1: Infection of RBCs with P. falciparum or P. knowlesi induces species-specific changes to the phosphoproteome of RBC proteins independently of blood type.
Fig. 2: Conditional knockout strategies for efficient excision and localization of the FIKKs.
Fig. 3: Each FIKK kinase influences the phosphorylation of distinct phosphosites on RBC and P. falciparum proteins.
Fig. 4: FIKK kinases are part of a complex network and act in synergy.
Fig. 5: FIKK4.1 is important for the modulation of iRBC rigidity and PfEMP1 surface translocation.

Data availability

The mass-spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE92 partner repository with the dataset identifier PXD015833. Gene sequences and annotations for P. falciparum 3D7 and P. knowlesi strain H were acquired from PlasmoDB.org (2018)72, and human sequences were acquired from Uniprot.org (2018). RNA sequencing data from Toenhake et al.37 was also used. Source data in the form of unprocessed gels and western blots corresponding to Figs. 2c, 3d and Extended Data Figs. 2,3 are available with the article.

Code availability

No custom code deemed central to the conclusions to this manuscript has been used in this study.

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Acknowledgements

We thank H. R. Saibil, P. Cicuta, M. J. Blackman and A. A. Holder for their critical feedback on the manuscript, and the members of the Treeck, Blackman and Holder labs for discussions throughout. We also thank B. Snijders and H. Flynn from the Mass-Spectrometry Science Technology Platform (MS-STP); A. Weston, M. Russell and L. Collinson from the Electron Microscopy STP; E. Christodoulou from the Structural Biology STP; N. O’Reilly, D. Joshi and H. Nagaraj from the Peptide Chemistry STP; and everyone from the Flow Cytometry STP at the Francis Crick Institute for their outstanding technical support and training. We thank J. Rayner and L. Parish for the MAHRP1 antibody, and the European Malaria Reagent Repository and J. McBride, University of Edinburgh, for the KAHRP antibody. We thank M. Jones for the initial experiments, and M. Koch and J. Baum from the Imperial College London for performing the initial flickering-analysis experiments. We also thank C. Lavazec for initial training for the microsphiltration experiments. This work was supported by funding from the Francis Crick Institute, which receives its core funding from Cancer Research UK, the UK Medical Research Council and the Wellcome Trust (grant nos FC001189 and FC001999 from all three funding bodies) and an Idea to Innovation Award (grant no. FC10550). V.I. is supported by the Engineering and Physical Sciences Research Council (EPSRC; grant no. EP/R011443/1) and a Sackler fellowship. M.K. receives support from the NIHR Imperial College BRC and the Wellcome Trust (Sir Henry Wellcome Fellowship grant no. 206508/Z/17/Z). C.B. was supported by an MRC Project Grant (grant no. MR/P010288/1). B.G., D.D.-S. and J.-P.S. are supported by the French National Research Agency (grant no. ANR-16-CE11-0014-01) and Laboratory of excellence GR-Ex, F-75015, Paris, France.

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Authors

Contributions

H.D. and H.B. performed all of the parasite genetic manipulations and phenotype analyses. H.D., H.B. and M.B. processed the proteomic samples. H.D., M.B., X.Y. and M.K. analysed the proteomic data. V.I. and H.B performed and analysed the flickering spectrometry. C.B. performed and analysed the electron tomography. D.D.-S. and J.-P.S. advised on the cytoadhesion experiments and performed the qPCR. M.Tibúrcio provided the NF54 DiCre-expressing parasites. H.D., H.B. and M.Treeck conceived the study, designed experiments and interpreted results. H.D., H.B., M.B., V.I., C.B., D.D.-S., J.-P.S. and M.Treeck wrote the original manuscript. H.D., H.B., M.B., V.I., C.B., D.D.-S., J.-P.S., B.G., M.K. and M.Treeck reviewed and edited the final manuscript.

Corresponding author

Correspondence to Moritz Treeck.

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Extended data

Extended Data Fig. 1 Statistical analysis of phosphorylation on RBC and P. falciparum proteins in different blood types.

(a) One way ANOVA test. (blood type) Testing the hypothesis that blood type does not affect RBC phosphorylation in infected or uninfected samples (A+=AB+=O+). F-value: 0.322 » 0.05, therefore blood type does not affect RBC phosphorylation. NS, not significant. N = 1 sample from each blood type. (b) Within-sample and species-specific testing of differences between blood types using one way ANOVA tests. Plots show phosphorylation intensity on uRBC proteins, log2 fold change on RBC proteins in iRBC-uRBC, and phosphorylation intensity on P. falciparum proteins, in two different blood types. There is no significant difference between blood types in any case (NS—not significant). Above each plot is the Pearson’s correlation coefficient (R) between the two blood types in the condition tested. There was no significant difference observed in any condition and there was a strong correlation between the phosphosite intensities in all blood types. **** - P ≤ 0.0001. N = 1 sample from each blood type.

Extended Data Fig. 2 Correct integration of the different FIKK cKO plasmids into the respective endogenous loci and excision of their kinase domains upon RAP treatment.

Schematics describing the different strategies used for generating the conditional FIKK knockout lines using selection linked integration or Cas9. Two LoxP introns were introduced on each side of the recodonised FIKK kinase domain, which was fused to a triple HA tag (red), a T2A skip peptide (blue) and a neomycin-resistance gene, to select for correct integration. Black arrows represent promoters and lollipops depict STOP codons. The relative positions of primers used to confirm correct integration of the plasmids into the respective loci and correct excision of the FIKK kinase domains upon RAP treatment are shown as coloured arrows. HR, homology region; Neo, neomycin-resistance cassette; RAP, rapamycin; rc. KD, recodonised kinase domain. Alongside the schematics are shown the PCR gels confirming correct integration and correct excision. DNA size markers in kbp are indicated on the left. PCRs for each FIKK were repeated at least 3 times with similar results for independent RAP-treatments. Source data

Extended Data Fig. 3 Integration of LoxP introns for the simultaneous excision of 7 FIKKs on chromosome 9 upon RAP treatment.

For deletion of all 7 FIKKs on chromosome 9, the entire locus was flanked with LoxP introns. A triple HA tag (red) was inserted into FIKK9.1. A T2A skip peptide (blue) and neomycin resistance gene allow selection for correct excision. Black arrows represent promoters and lollipops depict STOP codons. The relative positions of primers used to confirm correct integration of the plasmids into the respective loci and correct excision of the FIKK kinase domains upon RAP treatment are shown as coloured arrows. HR, homology region; Neo, neomycin-resistance cassette; RAP, rapamycin; rc. KD, recodonised kinase domain. Alongside the schematics are shown the PCR gels confirming correct integration and correct excision. DNA size markers in kbp are indicated on the left. PCRs were repeated at least 3 times with similar results. Source data

Extended Data Fig. 4 Exported FIKK kinases do not play a role in parasite growth under standard culture conditions.

(a and b) Parasite growth curves for Plasmodium falciparum NF54 (a) and 1G5 (b) FIKK conditional knockout lines. Starting parasitemia was adjusted to 0.5% and samples were fixed every 24 h for 120 h (excluding at 48 h for the 1G5 lines). Parasitemia was measured by flow cytometry on 3 biological replicates for all FIKKs, and the mean and SEM are shown. Statistical analysis by two way ANOVA with Tukey correction for multiple comparisons. (**** p ≤ 0.0001, * p ≤ 0.05, NS, not significant). Precise P values are shown in supplementary information table 2. (c) Scatter plots showing the area of plaques obtained by plaque assay for 1G5 FIKK conditional knockout lines, DMSO- (left) or RAP-treated (right). Horizontal bars indicate mean plaque area ± SD. Statistical significance was determined by a two-tailed t-test with no adjustment for multiple comparisons: 1G5 DMSO vs RAP (p = 0.1626); FIKK4.1 DMSO vs RAP (p = 0.4306); FIKK7.1 DMSO vs RAP (p = 0.7201); FIKK10.1 DMSO vs RAP (p = 0.5686); FIKK11 DMSO vs RAP (p = 0.7912); FIKK8 DMSO vs RAP (p = 0.0008) n = number of plaques, *** p < 0.001, NS, not significant.

Extended Data Fig. 5 FIKKs are localized to different subcellular compartments.

Localization pattern of additional FIKK kinases using antibodies against the C-terminal HA-tag fused to each FIKK kinase. DAPI was used for nuclear staining. The experiment was repeated at least 3 times independently for each FIKK with similar results. Scale bar - 5 µm. See also Fig. 2.

Extended Data Fig. 6 Unenriched proteome analysis of FIKK KO lines shows no difference in growth upon RAP-Treatment.

(a) Plots showing the Log2 Fold change in protein intensity (y axis) between FIKK4.1 DMSO and FIKK4.1 Rap (Upper panel) and between FIKK4.1 DMSO and FIKK12 DMSO (lower panel), against the total intensity for all samples. Protein intensity was calculated from the average reporter intensity of all peptides on a protein, and only proteins for which more than 1 peptide was detected were included. In red are all proteins transcribed 3x more at 40 hpi than at 35hpi, according to RNA seq data37. Between DMSO and RAP-treated lines the intensity of these proteins do not change substantially, while there is a clear increase in the FIKK4.1 DMSO line relative to FIKK12 DMSO, while all other proteins remain the same indicating a likely difference in growth. (b and c) To establish differences in growth between the lines, the log2 fold change in the intensity of the late-stage proteins between each line and the average of all lines within the experiment were calculated (b), or between RAP and DMSO-treated FIKK cKO lines (c). The log2 fold change for all other proteins was then subtracted from this to control for differences in protein abundance between samples.

Extended Data Fig. 7 FIKK-dependent species-specific RBC phosphorylation.

Phosphosites with the highest L2FC between P. falciparum and P. knowlesi (column 1) are labelled by any FIKK kinases which cause a significant reduction in phosphorylation of that site upon deletion (column 4).

Extended Data Fig. 8 Flickering analysis of FIKK4.1 knockout.

(a) Time-course flicker spectroscopy comparing the membrane bending modulus, the radius and the viscosity of uRBCs and RBCs infected with NF54::DiCre and FIKK4.1::DiCre DMSO- or RAP- treated parasites. Horizontal line within the box represents the median and whisker boundaries represent the 10th and 90th percentile. Points represent outliers. Statistical significance was determined by a two-tailed t-test. Precise p-values are included in supplementary information table 3. n = 2 biologically independent samples, the number of cells counted for each condition are summarized in supplementary information Table 1. * p < 0.05; NS, not significant. (b) Representative flickering spectra of DMSO- (in green) or RAP-treated (in blue) FIKK4.1::DiCre parasites at increasing time post-invasion. Mean square amplitude of fluctuations remains similar for DMSO-treated FIKK4.1::DiCre parasites throughout parasite development, while fluctuations in FIKK4.1 knockout parasites decrease significantly at 32 and 36 h post-invasion. Fitted modes 5–14. The error bars are calculated as \({\it{SD}}/\sqrt {\left( {{\it{n}} \times {\it{dt}}} \right)/{\it{\uptau }}_{{\it{q}}_{\boldsymbol{x}}}}\), where SD is the standard deviation, n total number of frames (□10000 frames per cell), dt time gap between each frame, and \({\it{\uptau }}_{{\it{q}}_x}\) the relaxation time for each mode. N = two biologically independent experiments.

Extended Data Fig. 9 Cytoadhesion and Var2CSA surface translocation of FIKK4.1 knockout.

(a) Var gene transcription profile of NF54::DiCre and FIKK4.1::DiCre parasites determined by qPCR. Transcriptional levels of each var genes were normalized with the housekeeping gene, seryl-tRNAtransferase. (b) SEM images of the surface of erythrocytes infected with DMSO or RAP-treated FIKK4.1::DiCre parasites. The experiment was repeated 2 times with similar results (scale bar - 1 µm). (c) Top panels: Electron micrographs of knobs on detergent treated, negatively-stained RBC ghosts from wild-type 3D7 and FIKK4.1KO schizonts imaged at low magnification. Red arrows indicate the position of knobs, which show up as circular dark patches on the membrane (scale bar - 200 nm). Bottom panels: Negative-stain electron tomography reveals typical structural features of the knob complex. In top views of knobs (XY), the knob coat is outlined with a red dashed line and the underlying knob spiral is indicated by red arrow heads. In these examples of knobs, the underlying spiral is left-handed (indicated by the blue spiral symbol) showing that the knob is pointing upwards from the plane of the grid. A side view (XZ) of the same knob shows that the height and diameter of the knobs in wild-type 3D7 schizonts FIKK 4.1KO schizonts is similar. Knobs with a right-handed spiral are shown in Fig. 5g. These knobs are pointing downwards and are compressed against the surface of the grid, hence more rings of the spiral structure are visible in the plane of the tomogram. Images are an average of 5 central slices of the tomogram (scale bar - 50 nm). (d) Gating strategy for microsphiltration flow cytometry experiments. (e) Gating strategy for Var2CSA surface expression experiments—the histograms shown in Fig. 5h include all infected cells, and the median PE- fluorescence is calculated on this population. A similar gating strategy was used for growth curves using Hoescht or Sybrgreen.

Extended Data Fig. 10 Correlation between the MS2 and MS3 mass-spectrometry methods.

Intensity of peptides which are observed by both methods (approximately 50% of all detected peptides), with MS2 intensities on the x axis and MS3 intensities on the y axis.

Supplementary information

Supplementary Information

Supplementary Discussion and Tables 1–3.

Reporting Summary

Supplementary Table 1

TMT quantification of phosphosites—raw data for all experiments.

Supplementary Table 2

Unenriched proteomes for FIKK KO datasets for analysing differences in growth.

Supplementary Table 3

Phosphoproteome of RBC infected with P. falciparum and P. knowlesi, and comparison of blood types (related to Fig. 1).

Supplementary Table 4

Phosphoproteome of FIKK knockout parasites (related to Fig. 3).

Supplementary Table 5

Phosphorylation motif for potential FIKK4.1 substrates (related to Fig. 5).

Supplementary Table 6

Primers and synthesized genes used for the generation of FIKK cKO lines, and primers used to confirm correct integration and excision upon RAP treatment.

Source data

Source Data Fig. 2

Unprocessed western blots.

Source Data Fig. 3

Unprocessed western blots.

Source Data Extended Data Fig. 2

Unprocessed gels.

Source Data Extended Data Fig. 3

Unprocessed gels.

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Davies, H., Belda, H., Broncel, M. et al. An exported kinase family mediates species-specific erythrocyte remodelling and virulence in human malaria. Nat Microbiol 5, 848–863 (2020). https://doi.org/10.1038/s41564-020-0702-4

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