Ubiquitin activation is essential for schizont maturation in Plasmodium falciparum blood-stage development

Abstract

Ubiquitylation is a common post translational modification of eukaryotic proteins and in the human malaria parasite, Plasmodium falciparum (Pf) overall ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite stages in the asexual blood stage cycle. Here, we identify specific ubiquitylation sites of protein substrates in three intraerythrocytic parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified (data available via ProteomeXchange with identifier PXD014998). 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, suggesting a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the apparent extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We created a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. Nuclear division and formation of intracellular structures was interrupted. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites.

Fig 1. Ubiquitylation throughout the parasite asexual blood stage.

(A) Ubiquitylation sites and their corresponding proteins detected in four stages of parasite development. (B) Distribution of detected ubiquitylated proteins between the intraerythrocytic ring, trophozoite and schizont stages and the extracellular merozoite stage. Whilst some proteins were found to be ubiquitylated in all four stages, the majority were found to be ubiquitylated only in merozoites. The primary data are presented in S1 and S3 Datasets. Maps depicting GO enrichment results for (C) the intra-erythrocytic parasite and (D) the merozoite ubiquitomes. The network representation of enriched GO terms reveals large clusters (A-I) of functionally-related GO terms. Nodes with border lines in blue represent GO terms that are found enriched in the ubiquitome of all stages. Identification of the GO terms and associated proteins is provided in S4 Dataset.

Fig 2. Treatment with MLN7243 arrests parasites at the schizont stage and blocks protein ubiquitylation.

(A) Parasites were treated with either MLN7243 or DMSO carrier, stained with Hoechst dye and analysed by FACS at three time points: 3, 44, and 64 h post invasion, shown in red, blue and orange respectively. Haploid ring stages transition to multinucleate schizonts (shown at 44 h) and in the control DMSO-treated cultures, merozoite reinvasion produces new haploid intraerythrocytic parasites (shown at 64 h). The MLN7243-treated parasites do not release merozoites or form new haploid intraerythrocytic parasites. (B) Microscopy of Giemsa-stained parasites at 24, 44 and 52 h post invasion, showing that both cultures develop into schizonts but by 52 h control parasites have formed new ring stages and the MLN7243-treated parasites remain as schizonts. (C) The percentage of parasitemia of the samples analysed in Panel A (mean +/- standard deviation of technical triplicates) in culture at 3, 44 and 64 h post infection measured by FACS analysis. During intracellular development there is no change in parasitemia, but by 64 h the parasitemia has increased in the control (DMSO-treated) and not the MLN7243-treated culture. (D) Protein ubiquitylation in schizonts either treated (+) or untreated (-) with MLN7243 during development from ring stages. Western blots were probed with a ubiquitin-specific antibody or antibodies specific for K48- and K63-linked polyubiquitylation. Antibodies to parasite GAPDH were used as a loading control. The size of molecular mass markers is shown on the left side of the panel.

Fig 3. Homology model of PfUBA1 in complex with ubiquitin and MLN7243.

(A) Overall structure of the ternary PfUBA1-ubiquitin-MLN7243 complex with α-helices drawn as cylinders and β-strands as arrows. Ubiquitin (Ub) is shown in yellow, PfUBA1 is coloured according to its domain architecture (AAD [purple] and IAD [teal]: active and inactive adenylation domains, HB [cyan]: helix bundle, FCCH [green] and SCCH [blue]: first and second catalytic cysteine half domains, UFD [red]: ubiquitin fold domain) and MLN7243 is shown in stick representation. (B) Chemical structure of MLN7243 (TAK-243). (C) Surface representations of ScUBA1 (left) and PfUBA1 (right) in the vicinity of MLN7243 which is coloured according to atom type with C-atoms in green, O-atoms in red, N-atoms in blue, S-atoms in yellow and F-atoms in light cyan. The locations of residues discussed in the text are indicated. MLN7243 is linked to ubiquitin via a covalent link to the terminal N-atom. (D) Differences in the residues surrounding the CF₃-group of MLN7243 are highlighted. HsUBA1 features the same residues indicated for ScUBA1, namely Pro554, Asp579 and Arg586.

Fig 4. Thioesterification of PfUBA1 E1 using fluorescently labelled ubiquitin (Ub) and the transthioesterification of four distinct E2s in vitro.

(A) In the presence of Pf UBA1, thioesterification of the E1 by Ub occurs in the absence of E2; in the presence of four distinct E2s (UBCH5, UBCH7, CDC34 and UBC13) further transthioesterification to the E2 occurs. No thioester formation is observed in the absence of ATP or after treatment with DTT. (B) Increasing concentrations of MLN7243 reduce covalent addition of ubiquitin to PfUBA1, allowing (C) the calculation of its IC₅₀. Data are shown as mean ± standard deviation from experiments performed on three different occasions. Expression, purification and further characterization of the recombinant PfUBA1 and additional control experiments are provided in S5 Fig.

Fig 5. Generation of a parasite line with a rapamycin-inducible uba1 gene knockout and its response to treatment with either rapamycin, MLN7243, or DMSO.

(A) CRISPR Cas9 was used to modify the 3' end of the uba1 gene in the II-3 parasite line, inserting loxPint in place of the last intron, a recodonised coding sequence fused to GFP, a second loxPint sequence and sequence coding for a triple HA tag. The parasite has a floxed gene from which a UBA1-GFP fusion protein is expressed. In the presence of rapamycin the floxed sequence is excised to produce an inactive truncated UBA1 protein. (B) Diagnostic PCR with primers designed to detect successful integration of the floxed sequence and its excision in the presence of rapamycin. DNA from the parental (II-3) and three cloned recombinant lines (G107, G109 and G111), following growth with or without rapamycin, was amplified using the primers 121 and HArev. Amplified bands of 1.8 kb and 0.74 kb were expected from the floxed and excised (+rapamycin) locus in the progeny parasites and no product was expected from the parental II-3. (C) Western blot with anti-GFP antibodies probed against schizont extracts of parasites (clones G107, G109 and G111) with the floxed and excised (+ rapamycin) uba1 locus. Reactivity of antibodies to BiP (Binding immunoglobulin Protein, GRP-78) was used as a loading control. (D) Growth of parental (II-3) and two independent recombinant parasite clones (G109 and G111) treated at the early ring-stage with either DMSO (control) or 20 nM rapamycin for 24 h and then stained with Hoechst dye and analysed by FACS 48 h later. The data are presented as the mean growth as a percentage of the DMSO-treated samples in triplicate samples from three independent experiments. (E) G109 parasites were treated at the early ring stage with either DMSO, rapamycin or MLN7243 and the parasitemia measured at specific time points after invasion following staining with Hoechst dye and FACS analysis. At 48h the parasites were washed to remove drug and culture was continued for a further 12 h. Data are shown as mean ± standard deviation of technical triplicates. Genetic truncation or inhibition of UBA1 blocked parasite growth. (F) G109 parasites were treated at the early ring stage with either DMSO, rapamycin or MLN7243 and the number of nuclei in individual parasites was counted at specific time points after invasion following staining with Hoechst dye and fluorescence microscopy. Shown is the median with the 95% confidence interval. One way ANOVA with Sidak’s multiple comparison test: **** DMSO v. RAPA, p < 0.0001, DMSO v. MLN7243, p < 0.0001; ^nsRAPA v. MLN7243 p = 0.937. (G) Immunofluorescence microscopy images of G109 parasites treated at the early ring stage with either DMSO or rapamycin and stained with antibodies to either GAP45 or ARO at 45 h after invasion. GAP45 is a component of the inner membrane complex (IMC) and ARO is a rhoptry protein. Merged images with DAPI staining and differential interference contrast (DIC) are included. Scale bar is 2 micron.