PfPPM2 signalling regulates asexual division and sexual conversion of human malaria parasite Plasmodium falciparum

Abstract

Malaria parasite undergoes interesting developmental transition in human and mosquito host. While it divides asynchronously in the erythrocytes, it switches to sexual forms, which is critical for disease transmission. We report a novel signalling pathway involving Protein Phosphatase PfPPM2, which regulates asexual division of Plasmodium falciparum as well as its conversion to sexual forms. PfPPM2 may regulate the phosphorylation of key proteins involved in chromatin remodelling and protein translation. One of the key PfPPM2-targets was Heterochromatin Protein 1 (HP1), a regulator of heritable gene silencing which contributes to both mitotic proliferation as well as sexual commitment of the parasite. PfPPM2 promotes sexual conversion by regulating the interaction between HP1, H3K9me3 and chromatin and it achieves this by dephosphorylating S33 of HP1. PfPPM2 also regulates protein synthesis in the parasite by repressing the phosphorylation of initiation factor eIF2α, which is likely to contribute to parasite division and possibly sexual differentiation.

Fig. 1. PfPPM2 regulates asexual division of Plasmodium falciparum.

A PfPPM2 has an insert between its PP domain and also has a putative N-terminal myristylation signal^,. In order to achieve conditional knockdown (cKD) of PfPPM2, a ribozyme glmS was introduced at its 3’end along with a HA-tag in 3D7 as well as NF54 background (Supplementary Fig. 1). The addition of glucosamine and subsequent conversion to GlcN6P activates the ribozyme and will result in its cleavage and degradation of the mRNA causing depletion of PfPPM2 protein. B Western blotting of PfPPM2-HA-glmS^3D7 parasites, which were either left untreated or treated with GlcN (cycle 0, rings) and parasite lysates were prepared (schizonts, cycle 1) using anti-HA antibody, which caused effective depletion of PfPPM2-HA. β-actin antibody was also used to probe the blots. Bottom Panel, Fold change in PfPPM2 depletion was determined by densitometry of PfPPM2 bands in the Western blots, which was normalized with respect to actin (Statistical differences were determined using paired Two-tailed Student’s t-tests, **P < 0.01. Single data points and mean ± SEM of three biological replicates are shown). C PfPPM2-HA-glmS^3D7(left panel) or PfPPM2-HA-glmS^NF54 (right panel) parasites were synchronized and ring stage parasites were used for setting up growth rate assay in the presence or absence of GlcN. Parasite growth was assessed after each cycle by performing flow cytometry (right panel) as well as analysis and counting parasites from Giemsa-stained smears (left panel). Inset, fold change in parasitemia of GlcN treated with respect to untreated parasites was determined (Statistical differences were determined using Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001. Single data points and mean ± SEM of four biological replicates are shown). The arrow indicates the time of GlcN-addition. D PfPPM2-HA-glmS^3D7/NF54 parasites were treated with GlcN as described in panel B. Thin blood smears were made at ~40–44 hpi in cycle 1. The numbers of nuclear centers/merozoites were counted from Giemsa-stained thin blood smears. (Statistical analysis was performed using unpaired Two-tailed Welch’s t-tests, ****P < 0.0001, n > 120 schizonts were counted in each condition. Each point represents a schizont and mean ± SEM of three biological replicates are shown). E PfPPM2-HA-glmS^NF54 parasites were synchronized and U-ExM was performed on schizonts during various stages of division (~ 36-44 h.p.i) using anti-HA and anti-tubulin antibody. The localization of PfPPM2-HA was mainly cytoplasmic early in division. However, in more mature schizonts it was frequently localized as puncta close to the nucleus. Scale bar =5 μm. A zoomed projection of individual merozoites is also provided. A projection of z-sections of images in rows 3 and 4 is also provided as videos in Supplement (Supplementary Movie 1 and 2). F PfPPM2-HA-glmS^NF54 parasites were treated with GlcN as described in panel B and C. U-ExM was performed on schizonts (cycle 1) using anti-tubulin and anti-centrin antibodies which revealed the presence of hemispindles, mitotic spindles and interpolar spindles in untreated parasites. GlcN treatment resulted in a loss of spindles in most parasites. Scale bar = 5 μm. Right Panel, percent parasites with normal tubulin staining- reflected by the presence of above-mentioned forms of spindles- were counted from U-ExM images (total number of parasites counted = 65 each in control and knockdown condition). The images for quantification were acquired from two independent experiments. Data is plotted as mean ± SEM of two biological replicates. Source data are provided as a Source Data file.

Fig. 2. PfPPM2 regulates the phosphorylation of key parasite proteins involved in chromatin remodelling and translation.

A PfPPM2-HA-glmS^3D7 parasites were treated with GlcN as described in Fig. 1B and C to perform comparative phosphoproteomic and proteomic analyses as indicated in the schematic (Supplementary Fig. 4). The S-curve represents fold-change ratios of identified phosphopeptides upon GlcN treatment (Supplementary Data 1.1). Some of the significantly altered phosphorylation sites (red and blue) belonging to key proteins relevant for protein synthesis or chromatin organisation (Supplementary Data 1.2, 1.3) are outlined in cyan and green, respectively. B Gene Ontology enrichment of proteins that exhibited significant changes in phosphorylation upon PfPPM2 depletion (Supplementary Data 1.5, 1.6). The phosphorylation fold-change ratio of identified phosphopeptides was normalized to total protein abundance fold-change. A one-sample, two-sided Student’s t-test was performed on the normalized values from three biological replicates (n = 3) to identify proteins with significant deviations. Pathways are represented based on their –log₁₀(P values), as estimated by the Gene Ontology tool available at PlasmoDB. C Sequence comparison of N-terminal tail of human histone H3 variants with P. falciparum Histone H3 and H3.3. Key motifs containing regulatory sites indicated by red square and critical residues that are modified by phosphorylation (S10, S28, S32), methylation (K9) and acetylation (K9, K27) are indicated. D Western blot analysis using specific antibodies against pS10-H3, pS28-H3 performed on NF54 and PfPPM2-HA-glmS^NF54 lines cultured in the absence and presence of GlcN. Lower Panel, densitometry analysis was performed and fold change in GlcN-treated parasites compared to untreated parasites was determined. (Statistical differences were determined using Two-way ANOVA, ** p < 0.01,**** p < 0.0001, P > 0.05,ns-nonsignificant. Single data points and mean ± SEM of three biological replicates are shown). Source data are provided as a Source Data file.

Fig. 3. PfPPM2 regulates HP1 phosphorylation and sexual conversion of the parasite.

A. PfPPM2 depletion causes hyperphosphorylation of S33 (*) present in its chromodomain (CD). S33 seems to be conserved in human HP1α and HP1β (not shown here) and is replaced by a D in S. pombe homologue swi6. Bottom panel, MS/MS spectra for peptide corresponding to pS33-HP1. B Western blotting of lysates from PfPPM2-HA-glmS^NF54 parasites, which were either left untreated or treated with GlcN using antibody against pS33-HP1 (see methods for details) or its phosphorylation-independent version. Bottom Panel, Fold change in HP1-S33 phosphorylation upon PfPPM2 depletion in experiments described in the upper panel was determined by densitometry (Statistical differences were determined using unpaired Two-tailed Student’s t-tests, *P < 0.05. Single data points and mean ± SEM of four biological replicates are shown). C PfPPM2-HA-glmS^NF54 or NF54 (control) parasites were cultured in the presence or absence of GlcN and gametocyte formation was induced as described in Methods. The number of gametocytes formed after GlcN-treatment was counted and fold change in sexual conversion and % conversion of asexual parasites to gametocytes was determined at day 5 p.g.i. (Statistical analysis was performed using Two-way ANOVA, **** p < 0.0001; **P < 0.01, ns p > 0.05. Single data points and mean ± SEM of four biological replicates are shown). D Gametocyte formation was induced in GlcN-treated or untreated PfPPM2-HA-glmS^NF54 parasite. qRT-PCR was performed after 24 h of gametocyte induction to compare the expression of AP2-G and HP1 (Statistical analysis was performed using paired Two-tailed Student’s t-tests, *p < 0.05, ns p > 0.05). Single data points and mean ± SEM of three biological replicates are shown). E ChIP-qPCR was performed to determine PfHP1 and H3K9me3 occupancy at AP2-G or Pfs16 loci in PfPPM2-HA-glmS^NF54 parasites which were either left untreated or treated with GlcN. IgG antibody was used for ChIP as a negative control (Statistical analysis was performed using paired Two-tailed Student’s t-tests, **P < 0.01, *p < 0.05, ns p > 0.05. Single data points and mean ± SEM of three biological replicates are shown). F Western blotting of lysates from PfPPM2-HA-glmS^NF54 or NF54 parasites, which were either left untreated or treated with GlcN using antibodies against H3K9me3, H3K9ac or total H3. Bottom Panel, Fold change in H3K9me3 and H3K9ac upon PfPPM2 depletion in experiments described in the above panel was determined by densitometry (Statistical analysis was performed using Two-way ANOVA, **P < 0.01, ns p > 0.05. Single data points and mean ± SEM of three biological replicates are shown). Source data are provided as a Source Data file.

Fig. 4. PfPPM2 mediated HP1 phosphorylation may be critical for asexual development and sexual differentiation.

A PfPPM2-HA-GlmS^NF54 parasites or HP1-GFP overexpressing PfPPM2-HA-glmS^Nf54:HP1-GFP^OE parasites were synchronized, treated with GlcN (cycle 0) and growth rate assays were performed to determine the parasitemia by flow cytometry as described for Fig. 1C. Inset, fold change in parasitemia was determined with respect to untreated parasites in the next two cycles (Statistical analysis was performed using Two-way ANOVA, **P < 0.01, ***P < 0.001, P > 0.05, ns-non-significant. Single data points and mean ± SEM of three biological replicates are shown). B PfPPM2-HA-GlmS^NF54 or PfPPM2-HA-glmS^Nf54:HP1-GFP^OE parasites were treated with GlcN as described in panel A. Thin blood smears were made ~40–44 hpi in cycle 1. The number of nuclear centers/merozoites were counted from Giemsa-stained thin blood smears. (Statistical analysis was performed using one-way ANOVA, ****P < 0.0001, ***P < 0.001, ns-nonsignificant, n  > 100 schizonts were counted in each condition and each data point represents a schizont. Mean ± SEM of three biological replicates are shown). C IFA was performed on PfPPM2-3xHA-GlmS^{NF54: HP1/HP1-S33A-Flag} parasites in which WT-HP1 or its S33A mutant (Supplementary Fig. 7) were tagged with Flag. Anti-Flag antibody was used to detect parasites with tagged proteins and anti-Pfs16 was used to stain gametocytes. Scale bar =2 μm. A representative image of Giemsa-stained smear of S33A mutant parasites with a significant number of gametocytes (red arrows). Bottom panel, % Flag-tagged parasites that exhibited Pfs16 expression in IFA. (Statistical analysis was performed using paired Two-tailed Student’s t-test, **, P < 0.01. n  > 120 parasites each in WT-HP1 and S33A-HP1 were counted. Single data points and mean ± SEM of three biological replicates are shown). Source data are provided as a Source Data file.

Fig. 5. Phosphorylation state of HP1 at S33 is critical for asexual and sexual development of P. falciparum.

A. HP1/S33A/S33D-GFP-DD parasites-which were generated by overexpressing HP1 or its S33A/D mutants with a GFP and DD domain (Supplementary Fig. 8A,B) were synchronized and ring stage parasites were used for setting up growth rate assay in the presence or absence of Shld-1. Parasitemia was determined after each cycle at the indicated time points from Giemsa-stained thin blood smears of parasite cultures and are represented as % of total number of parasites (Statistical analysis was performed to compare growth of shld1-treated S33A (orange) and S33D (grey) mutant with that of WT-HP1(blue) over expressing parasites using Two–way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are mean ± SEM of three biological replicates). B Thin blood smears were made for HP1/S33A/S33D-GFP-DD parasites cultured in the presence of Shld1 at indicated time point and stained with Giemsa to assess their morphology. Gametocytes dominated S33A cultures from cycle 1 and in the case of S33D mutant parasites several parasites exhibited abnormal morphology (red arrows). Bottom Panel, % parasites with abnormal or gametocyte morphology was determined. Single data points and mean ± SEM of two biological replicates are shown. C IFA was performed on thin blood smears of HP1/S33A/S33D-GFP-DD parasites cultured in the presence of Shld1 using anti-GFP and anti-Pfs16 antibodies. Right Panel, % of GFP positive parasites that exhibited Pfs16 staining was determined (Statistical analysis was performed using One-way ANOVA, **P < 0.01, ns- p > 0.05. n > 90 parasites each in HP1/S33A/S33D were counted. Single data points and mean ± SEM of three biological replicates are shown). Scale bar = 2 μm. D qRT-PCR was performed on Shld-1 treated HP1/S33A/S33D-GFP-DD parasites in cycle 1 to assess the fold change in AP2-G and Pfs16 expression with respect to WT-HP1 overexpressing parasites (Statistical analysis was performed using One-way ANOVA, *P < 0.05, p > 0.05 ns-not significant. Single data points and mean ± SEM of three biological replicates are shown). E. Nuclear protein lysates prepared from NF54 or HP1/S33A/S33D-GFP-DD parasites cultured in the presence of Shld-1 were used for immunoprecipitation using anti-GFP antibody. The IP (upper panel) or protein lysate (bottom panel) were used for Western blotting using indicated anti-GFP or anti-H3K9me3 and anti-H3 antibodies. Upper Right Panel, H3K9me3 co-immunopreciptated with HP1 or S33A/S33D mutants was quantitated by densitometry after normalization with respect to H3K9me3 present in total lysate (Bottom Panel). Subsequently, fold change in H3K9me3 present in IP of S33A/S33D mutants with respect to WT HP1 was determined. (Statistical analysis was performed using One-way ANOVA, **P < 0.01, p > 0.05, ns. Single data points and mean ± SEM of four biological replicates are shown). Bottom Right Panel, Fold change in H3K9me3 levels was determined in S33A/D mutants with respect to WT HP1 after normalization with respect to total H3 (Statistical analysis was performed using One-way ANOVA,**P < 0.01, P > 0.05, ns. Single data points and mean ± SEM of four biological replicates are shown). Source data are provided as a Source Data file.

Fig. 6. Dephosphorylation of HP1 is a novel signal for sexual differentiation of the parasite.

A IFA was performed on thin blood smears of HP1/S33A/S33D-GFP-DD parasites cultured in the presence of Shld-1 using anti-pS33-HP1 and anti-Pfs230 antibodies, revealed that pS33-HP1 was present mainly in the asexual stages (HP1/HP1S33D) but it was absent from HP1-S33A parasites, majority of which were gametocytes (Statistical analysis was performed using One-way ANOVA, ***P < 0.001, p > 0.05 ns-non-significant, n  > 120 parasites were counted in each condition. Single data points and mean ± SEM of three biological replicates are shown). Scale bar = 2 μm. B Sexual conversion of PfPPM2-HA-glmS^NF54 cultured in the absence or presence of GlcN was induced by serum depletion as indicated in the schematic. IFA was performed on parasites pre- and post-induction using anti-pS33-HP1 and anti-Pfs230 antibodies. % pS33-HP1 positive parasites in each condition were determined (n  > 80 parasites were counted in each condition. Single data points and mean ± SEM of two biological replicates are shown). Scale bar = 2 μm. C Schematic illustrates a novel signalling pathway in P. falciparum. Present studies demonstrate that PfPPM2 is important for both asexual division and sexual differentiation of P. falciparum. A yet to be identified kinase phosphorylates HP1 at S33, which is important for asexual development and division. S33-HP1 phosphorylation promotes its association with H3K9me3 and maintenance of heterochromatin state is critical for asexual division. PfPPM2 mediated dephosphorylation of HP1 at S33 prevents the interaction of HP1 with H3K9me3 and promotes euchromatin formation and derepression of AP2-G, which is critical for sexual differentiation. Created in BioRender. Sharma, P. (2025) https://BioRender.com/arwn5m3. Source data are provided as a Source Data file.