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. 2022 Aug 4;13(1):4537.
doi: 10.1038/s41467-022-32271-7.

Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X

Sumit Mukherjee  1 , Suong Nguyen  1 , Eashan Sharma  1 , Daniel E Goldberg  2
Affiliations

Maturation and substrate processing topography of the Plasmodium falciparum invasion/egress protease plasmepsin X

Sumit Mukherjee et al. Nat Commun. .

Abstract

The malaria parasite Plasmodium invades a host erythrocyte, multiplies within a parasitophorous vacuole (PV) and then ruptures the PV and erythrocyte membranes in a process known as egress. Both egress and invasion are controlled by effector proteins discharged from specialized secretory organelles. The aspartic protease plasmepsin X (PM X) regulates activity for many of these effectors, but it is unclear how PM X accesses its diverse substrates that reside in different organelles. PM X also autoprocesses to generate different isoforms. The function of this processing is not understood. We have mapped the self-cleavage sites and have constructed parasites with cleavage site mutations. Surprisingly, a quadruple mutant that remains full-length retains in vitro activity, is trafficked normally, and supports normal egress, invasion and parasite growth. The N-terminal half of the prodomain stays bound to the catalytic domain even after processing and is required for proper intracellular trafficking of PM X. We find that this enzyme cleaves microneme and exoneme substrates before discharge, while the rhoptry substrates that are dependent on PM X activity are cleaved after exoneme discharge into the PV. The data give insight into the temporal, spatial and biochemical control of this unusual but important aspartic protease.

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Conflict of interest statement

All authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Autoprocessing sites are conserved between the heterologously expressed and parasite PM X.
a Schematic of the likely identities of the different processed forms of C-terminally 8x His tagged PM X (rPM X) expressed from the HEK293 cells. Predicted mass of the polypeptides based on their amino acid compositions are indicated. Thick lines indicate the processed fragments that were detected either by western blot or by Coomassie blot. Orange thin lines represent processed bands not detected. Amino acid sequence flanking the PM X autocleavage sites are indicated with positional information. Green dotted arrows represent the scissile bonds. PD prodomain, CAT catalytic domain. The red, blue and gray arrows represent the peptide coverage regions obtained from LC/MS. Filled arrow heads: tryptic ends, empty arrow heads: nontryptic ends. Colors correspond to the bands highlighted by same color arrows in b and d. Additional mutated constructs used in this study are depicted in Supplementary Fig. 1. b Left, anti-His immunoblot; right, Coomassie stain of wild type rPM X (rWT). Anti-His immunoblot (c) and Coomassie gel (d) showing processing of rWT or mutant rPM X at multiple autocleavage sites. rSS/IADbl mut : SSAA/IAAA double mutant; rQuadmut : quadruple mutant with additional NFAA and SDAA mutations. e Expression of second-copy PM X in parasites. Left, schematic of the second copy PM X that was introduced into parasites as a C-terminally GFP-tagged construct. Labels as in a. Right, top panel: anti-GFP immunoblot, the assigned unprocessed and differentially processed forms indicated by color coded lines. Bottom panel: lysates from the same samples but blotted with anti-PM V antibody as loading control. NF/SDDbl mut : NFAA/SDAA double mutant; SS/IADbl mut : SSAA/IAAA double mutant; Quadmut : quadruple mutant with all four cleavage sites changed to AA in the P1’ and P2’ positions. Each experiment (b–e) was repeated at least three times and shown are representative blots. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Autocatalytic processing of PM X is not required for parasite replication and substrate cleavage.
a The parental PM Xapt parasites or PM Xapt parasites expressing the indicated PM X as a second copy were grown either in the presence or absence of aTc for 96 h. The starting parasitemia was 1% across different samples. Shown are the final parasitemia percentages after 2 erythrocytic cycles, normalized to that of the PM Xapt parasites grown in the presence of aTc for 96 h. Mean values from three independent experiments are shown and error bars represent standard deviations. Data were analyzed statistically by two-tailed Student’s t test. ***p = 0.00002. b The parasite lines from a were MACS synchronized for 3 h and were grown for the next 45 h either in the presence or absence of aTc. Parasites were then harvested and whole cell lysates were prepared. Western blots were performed to detect the expression of the second copy PM X-GFP (top panel), endogenous PM X-FLAG (second panel), AMA1 (third panel) and SUB1 (bottom panel). On the right is the PM Xapt line without second-copy PM X expression. The antibodies used and the specific molecular weights of the different processed forms of proteins are indicated. This experiment was repeated two times and shown is a representative blot. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Proteolytic processing is not necessary for in vitro activity of PM X.
Second copy PM X-GFP was purified from synchronized 42–45 h schizonts using anti-GFP antibody. The pulled down proteins were then incubated with fluorogenic Rh2N substrate peptide (1 μM) at the indicated pH. In control wells, CWHM-117 (1 μM) was added to inhibit PM X activity. Reactions were carried out for 1 h at 37 °C. Mean values from three independent experiments are shown and error bars represent standard deviations. Data were analyzed statistically by two-tailed Student’s t test, p values are shown on the graph. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. The p12 polypeptide remains attached to rPM X under activation conditions.
a Overlay of the elution profiles of WT rPM X (top) or hemoglobin a (bottom) following size exclusion chromatography at the indicated pH. b Elution profile of rPM X following size exclusion chromatography at pH 5.5 in the absence (top) or presence (bottom) of 6 M guanidine HCl (GuHCl) as a denaturant. Samples were pretreated in the same buffer before loading onto the column. Numbers within dotted lines indicate the fractions that were collected to run on SDS-PAGE followed by Coomassie staining (c). Subfractionation analysis of the pH 5.5 peak is shown in Supplementary Fig 5. d Comparison of the molar ellipticity of WT rPM X preincubated at the indicated pH and analyzed by circular dichroism spectroscopy. Experiment was repeated three times in triplicate. Shown are the mean values from one representative experiment. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. The N-terminal half of the prodomain (PD) is critical for intracellular trafficking and functionality of PM X.
a Scheme of PD truncation constructs that are C-terminally GFP tagged and introduced into the PM Xapt line as second copies. b Representative 2D confocal Airyscan images showing colocalization between the second copy PM X and either SUB1-3xHA or PM V. Scale bar: 2 μm. The 3D version of the images are shown in Supplementary Fig. 7. c Quantification from Supplementary Fig. 7. Experiments were repeated two times, and for each line 10 schizonts were analyzed. Mean values are shown. Error bars represent standard deviation. ***p < 0.001. For exact p values refer to the source file. d Zaprinast-induced discharge of second copy WT and delB but not delA mutant PM X from 41–44 h schizonts. Samples were treated with 75 μM zaprinast for 45 min. Supernatant was then separated from the cellular pellet followed by western blots with indicated antibodies. Shown are the representative blots from two independent experiments. e Growth curve of indicated parasite lines showing that the delA and delPD mutants failed to rescue the growth defect due to knockdown of PM X. Mean values from three independent experiments are shown and error bars represent standard deviations. For c and e data were analyzed statistically by two-tailed Student’s t test. P values in e represent comparison of delA mutant grown in presence or absence of aTc at two time points. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. PM X is processed in terminal secretory compartments in the parasites.
Synchronized 40–43 h schizonts expressing PM X-3xHA were treated separately with the vehicle (DMSO), E64d (10 μM) and C1 (1.5 μM) for 8 h. Samples were fractionated to separate the secreted (supernatant) components from those that remained intracellular (merozoites). PM X-3xHA was pulled down using anti-HA antibody. Western blot was done with both fractions to assess distribution of PM X. Aliquots were blotted for PM V as an intracellular control. Shown are representative blots from two independent experiments. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. PM X cleaves microneme substrates intracellularly, while rhoptry substrate cleavage is mediated in a post-secretion manner.
a, b PM Xapt parasites were grown in presence or absence of aTc to the schizont stage. At 44 h post invasion, either C1 (1.5 μM) or E64d (10 μM) was added to the +aTc cultures for 6 h before harvesting. Samples were blotted assess the cleavage of either AMA1 (a microneme substrate) or Rh5 (b rhoptry substrate). Processing of SUB1 and SERA5 were analyzed from the same lysates as indicators of PM X activity and exoneme discharge respectively. Each blot was repeated at least twice. Source data are provided as a Source Data file.
Fig. 8
Fig. 8. PM X and SUB1 signals overlap significantly more with microneme markers (AMA1 and EBA175) than with rhoptry neck (RON4) or bulb (RAP1) markers in terminal schizonts.
Synchronized, C1-treated 48–50 h schizonts expressing either PM X-3xHA or SUB1-3xHA were fixed in paraformaldehyde and processed for immunofluorescence assays. After labeling with the indicated antibodies, samples were visualized by confocal Airyscan microscopy. Images in a are 2D snapshots. 3D reconstructions are shown in b. Each grid line in b is 1.61 μm. Shown are representative images from two independent experiments. c Quantification of the 3D images from b. For each line, 10 schizonts were analyzed from three biological replicates. Mean values are shown and error bars represent standard deviations. Data were analyzed statistically by two-tailed Student’s t test, p values are shown on the graphs. Source data are provided as a Source Data file.
Fig. 9
Fig. 9. A subset of the apically located vesicles in schizonts show colocalization between AMA1 and PM X or SUB1 by immunoEM.
Synchronized, C1-treated, 48–50 h schizonts expressing either PM X-3xHA (a) or SUB1-3xHA (b) were processed as described in the methods. Thin section samples were labeled for the indicated markers and visualized by immunoelectron microscopy. Black arrows: 18 nm beads that detect the anti HA antibody, yellow arrows: 12 nm beads that detect the anti AMA1 antibody. Sections showing apical vesicles are magnified from each image. R rhoptry, N nucleus. Shown are representative images from two independent experiments.
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