A novel micronemal protein MP38 is involved in the invasion of merozoites into erythrocytes

Abstract

The absence of an in vitro cultivation system for Plasmodium vivax hinders the exploration of molecular targets for vaccine development. To address this, functional studies often rely on alternative models, such as P. knowlesi, due to its genetic similarity to P. vivax. This study investigated the role of a novel micronemal protein, PvMP38 (PVX_110945), in both P. vivax and P. knowlesi merozoite invasion of erythrocytes. The full-length ectodomain of PvMP38 was expressed, and polyclonal antibodies were generated to assess its function. PvMP38 was confirmed to localize on micronemal organelle in both P. vivax and P. knowlesi merozoites. In vitro protein-protein interaction assays revealed that PvMP38 binds to Pv12 with high-affinity interaction. A conserved novel complex of Pv12-Pv41-PvMP38 was identified by immunoprecipitation of P. vivax antibodies on P. knowlesi schizont lysates. Linear epitopes of PvMP38 with high and moderate antigenicity were identified in clinical isolates of both species. Invasion inhibition assays demonstrated that a triple antibody combination targeting the PvMP38, Pv12, and Pv41 significantly reduced P. knowlesi merozoite invasion of erythrocytes compared to a single antibody. In addition, CRISPR/Cas9-mediated knockout of P. knowlesi mp38 markedly impaired parasite growth, underscoring its essential role during the asexual stage. These findings identify PvMP38 and its associated complex as promising targets for malaria interventions and highlight the utility of P. knowlesi as a model for investigating P. vivax erythrocyte invasion mechanisms.IMPORTANCEThis manuscript reported an effort in malaria eradication by identifying and functionally characterizing a novel Plasmodium vivax micronemal protein, PvMP38, involved in erythrocyte invasion. A narrow repertoire of an efficacious vaccine targeting P. vivax candidates is being developed due to the lack of continuous in vitro culture. This study addresses a gap in P. vivax research using P. knowlesi as a model for both genome editing and antibody functionality validation. By enhancing the protein-protein interaction screening framework, this study demonstrated that PvMP38 forms a complex with Pv12 and Pv41, opening the approaches to multi-antigen vaccines. The successful application of CRISPR/Cas9 gene editing techniques to disrupt its homolog, the pkmp38 gene, further assesses the protein's significance in the growth and invasion of the parasite. These findings provided valuable insights into the biology of P. vivax and proposed PvMP38 as a promising candidate for malaria intervention strategies.

Keywords: CRISPR/Cas9; Plasmodium vivax; PvMP38; complex formation; malaria; micronemal protein.

Fig 1

Recombinant protein expression. (A) Schematic structure of target proteins. (B) Amino acid sequence alignment of PVX_110945 (PvMP38) with orthologs in P. knowlesi, PkA1H_060026500 (PkMP38), the similarity is highlighted in red. The percent identity and divergence were analyzed using Clustal-W. (C) Recombinant PvMP38 (~37.8 kDa with 8× His tag) under reducing (R) and native condition (N). (D) Western blot analysis of recombinant PvMP38 probed with anti-His tag antibody for the positive control (lane 1), mouse- (lane 2), and rabbit-immune sera (lane 3). Recognition of the native PvMP38 antigen in the P. knowlesi schizont parasite lysate with mouse and rabbit antisera raised against the recombinant PvMP38 under reducing conditions (lanes 4 and 5) and the specificity confirmation by pre-immune mouse and rabbit sera (lanes 6 and 7). For protein-protein interaction analysis, other proteins were expressed with His-tag (E) and Fc-tag (F). Pv12 and Pv41 were expressed under His-tag (E, lanes 1 and 2) and were fused with Fc-tag (F, lanes 1 and 2). For positive and negative controls of protein-protein interaction analysis, PvDBP-RII-His (E, lane 3), human DARC-Fc (F, lane 3), and GST under His- or Fc-tags (E and F, lanes 4) were also expressed, respectively.

Fig 2

Identification and quantification of the protein-protein interactions. (A) Analysis of the interaction between Fc- and His-tagged recombinant protein by ELISA. (B) A line graph represents the ELISA absorbance of panel A; data points represent the means of two technical replicates, and error bars represent SD. (C–H) The recombinant His-tagged protein of each testing pair was injected over immobilized Fc-tagged recombinant protein. Raw data from two technical replicates of BLI experiments with different batches of protein in Table S2.

Fig 3

Subcellular localization of anti-PvMP38 with organelle-specific antibodies in the schizont stage of P. vivax (A)- and P. knowlesi (B)-infected parasites by immunofluorescence assay. (A) Reactivity of PvMP38 mouse polyclonal antibody (red) to P. vivax schizont-stage parasites co-localized with P. vivax surface protein, PvMSP1-19, microneme protein, PvDBP-II, rhoptry body (PvRAMA) and rhoptry neck protein (PvRON2) (each in green). (B) Cross-reactivity of mouse anti-PvMP38 (red) to P. knowlesi A1-H.1 schizont-stage parasites as red color co-localized with P. knowlesi surface protein, PkMSP1-19, microneme protein, PkDBPα-II, rhoptry body (PkRAMA) and rhoptry neck protein (PkRON2) (each in green). All samples were counterstained with antibodies raised from rabbits. (C) P. vivax and P. knowlesi, and (D) E64-treated P. knowlesi parasites were dual-labeled with rabbit antisera against Pv12/Pv41 (green) and mouse anti-PvMP38 (red). “Inset” panels depict enlarged regions. Non-specific staining is detected using PvMP38 pre-immune serum, proving the specificity of the produced antibody. Bars represent 5 mm. DAPI, 4',6'-diamidino-2-phenylindole.

Fig 4

Disruption of pkmp38, an orthologue of pvmp38, in P. knowlesi parasites. (A) The donor DNA fragment was co-transfected with the pCas/sg construct that confers Cas9 nuclease, sgRNA, and drug resistance for further selection to knock out the pkmp38 gene by double cross-over homologous recombination. The yellow box represents the barcode gene absent in the P. knowlesi genome for genotyping. Small black arrows indicate the primers. (B) Genotyping of parasite cloned out by limiting dilution. Target and disrupted fragments shown in panel A were amplified with primer pairs indicated in the box. TF, transfected parasites; WT, wild type; P, housekeeping gene. (C) Western blot analysis of WT and KO parasite lysates. Anti-PvMP38 recognized native antigens in P. knowlesi WT but not in KO parasite lysates. Non-immune rabbit IgG was used as a negative control. (D) Co-localization in P. knowlesi A1-H.1 (WT) and Pkmp38^-(KO) using polyclonal antibodies against PvMP38. Rabbit anti-PkDBP antibody (green) was dual labeled with mouse anti-PvMP38 antibody as a localization marker. (E and F) Parasitemia and multiplication rate at 10 cycles of P. knowlesi WT and KO were measured. After recovery from the knockout process, the initial parasitemia was diluted to less than 0.1% and followed up for 10 days. Significant differences in the effects of pre-immune sera and other antibodies were calculated using an unpaired t-test, ns, not significantly different P > 0.05; *, P < 0.05.

Fig 5

Co-immunoprecipitation assay using rabbit polyclonal antibodies (Co-IP Ab). (A) Total protein lysate from wild-type and knockout parasites were extracted and separated by 13% SDS-PAGE under reducing conditions, and followed by silver staining. (B) Specificity of mouse anti-Pv12, anti-Pv41, and anti-PvMP38 antibodies to recognize their orthologs in P. knowlesi by western blot analyses. (C) Western blot analyses were performed using mouse-raised antibodies to detect immunoprecipitated antigens (IA) in wild-type (lane 1) and knockout (lane 2) parasite, pulled down by rabbit sera against Pv12 (RaPv12), Pv41 (RaPv41), or PvMP38 (RaPvMP38). Normal rabbit IgG was used as a negative control for the co-immunoprecipitation assay.

Fig 6

Humoral response of heat-treated and native PvMP38 to knowlesi (K) and vivax (V) patient sera. Normalized MFI represents the mean fluorescence intensity (MFI) divided by the cutoff value (equal to the mean fluorescence intensity plus two standard deviations (SDs) of the malaria-naïve samples). The bar indicates the mean ± standard deviation. Individuals with outlier reactivity were indicated in the black dot. The prevalence of antibody response was compared to the patients and healthy individuals (H) using the multi-variant one-way ANOVA and Tukey’s secondary test, ns, not significantly different P > 0.05; *P < 0.05; **P < 0.001; ***P < 0.0001.

Fig 7

The invasion-inhibitory activity of P. vivax antibodies in single (A) and combination (B) against P. knowlesi A1-H.1. (A) Total IgGs purified from rabbit sera were tested individually for invasion-inhibitory activities (0.66 to 4.0 mg/mL). (B) Combinations of two IgGs were assessed at two concentrations (2.0 and 2.0 mg/mL, and 1.0 and 1.0 mg/mL), and combinations of three IgGs were tested at 0.33, 0.66, and 1.33 mg/mL each. 2C3 IgG (25 µg/mL) was used as a positive control. To determine whether the inhibition was statistically significant, all treatments were independently compared to the same concentration of total IgG of the negative control, which is the inhibition of purified IgG from pre-immune rabbit sera (single) combined with GST-his rabbit immunized (combination). Two independent assays were performed in duplicate. Significant differences in the effects of pre-immune sera and other antibodies were calculated using a multi-variant one-way ANOVA and Tukey’s secondary test, ^*/#P < 0.05; **^/##P < 0.01; ***^/###P < 0.001. * represents the difference among groups within the graph. # represents the difference between single and combination antibody strategies, where 2 + 2 and 1 + 1 double combination sets (2 mg/mL and 1 mg/mL of each antibody) were compared with 4 mg/mL and 2 mg/mL of its single antibody components. A triple combination set of 1.33 + 1.33 + 1.33, 0.6 mg/mL, and 0.3 mg/mL each were compared with three single antibodies at 4, 2, and 1 mg/mL.