An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies
- Research Centre for Infectious Diseases, School of Biological Sciences, Adelaide University, Australia
- Department of Biochemistry and Chemistry, La Trobe Institute for Molecular Sciences, La Trobe University, Australia
- Institute for Photonics and Advanced Sensing (IPAS), Adelaide University, Australia
- AdAlta, Australia
- Burnet Institute, Australia
- Department of Infectious Diseases, University of Melbourne, Australia
- School of Translational Medicine and Department of Microbiology, Monash University, Australia
Figures
Figure 1 with 4 supplements
Presence and structure of MSP2 across different Plasmodium species.
(A) Schematic of the gene arrangement surrounding msp2 on P. falciparum chromosome 2. (B) AlphaFold2 structural predictions of example Laverania (P. falciparum [3D7], P. reichenowi, P. billcollinsi, P. adleri) and the two avian (P. relictum, P. gallinaceum) malaria species. The N-terminal signal peptide and C-terminal GPI anchor sequences were removed before the protein structure was predicted. The most N-terminal amino acid is indicated. Colours represent the predicted local distance difference test (pLDDT) scores, with dark blue representing very high confidence (>90%), light blue high confidence (90 to >70), yellow low confidence (70–50), and orange very low confidence (<50). An enlarged modelled structure of the N-terminal helical region is provided for P. falciparum 3D7 MSP2 on the left of the panel (grey shading), and also for the other examples of Laverania and avian malaria merozoite surface proteins (MSPs) (various colours) superimposed on the structure of P. falciparum 3D7 MSP2 (green). (C) Maximum likelihood tree showing relationship of MSP2 protein sequences found in different P. falciparum isolates and other Plasmodium species. Grouping of sequences from P. falciparum isolates into two main allele types can be seen as well as the separation of the Laverania and avian malaria species into their expected groups. Tree robustness tested by bootstrap.
Figure 1—figure supplement 1
Alignment of full-length amino acid protein sequences for all PfMSP2-like sequences on PlasmoDB.
Alignment done using MUSCLE alignment with default settings. Amino acids bound by red box indicate putative N-terminal signal peptide while those bound by a black box indicate putative GPI signal sequence.
Figure 1—figure supplement 2
Alignment and Amino Acid properties of the N-terminal conserved region of MSP2.
(A) Alignment (Blosum 62) of amino acid sequences for the N-terminal conserved region of P. falciparum 3D7, available Laverania, P. relictum, and P. gallinaceum MSP2-like sequences on PlasmoDB and identified through a BLAST search on the NCBI server. P. sp. 1, 2, and 3 represent MSP2 sequences identified using a BLAST search that are indicated to be from an unspecified Laverania spp. parasite. Amino acids are coloured according to their properties (RasMol). (B) Amino acid sequence properties for the N-terminal conserved region of P. falciparum 3D7, other Laverania species (including three Laverania sequences of unknown speciation), P. relictum and P. gallinaceum MSP2.
Figure 1—figure supplement 3
Alignment and Amino Acid properties of the C-terminal conserved region of MSP2.
(A) Alignment of amino acid sequences for the region corresponding to the C-terminal conserved region of P. falciparum 3D7, available Laverania, P. relictum, and P. gallinaceum MSP2-like sequences on PlasmoDB and identified through a BLAST search on the NCBI server. P. sp. 1, 2, and 3 represent MSP2 sequences identified using a BLAST search that are indicated to be from an unspecified Laverania spp. parasite. Initial alignment done using Blosum62 with default settings, with subsequent manual modification as required to align to the cysteine residues and other prominent areas of conservation between the Laverania and avian malarias. Amino acids are coloured according to their properties (RasMol). Insertions of 20 (P. relictum) and 26 (P. gallinaceum) amino acids are evident for the avian malarias, and P. relictum has only one of the two C-terminal cysteine residues that are prominent in the other aligned MSP2s. (B) Amino acid sequence properties for the C-terminal conserved region of P. falciparum 3D7, other Laverania species (including three Laverania sequences of unknown speciation), P. relictum and P. gallinaceum MSP2.
Figure 1—figure supplement 4
Alignment and Amino Acid properties of the N-terminal repeat region of MSP2.
(A) Amino acid sequences for the N-terminal repeat region of P. falciparum 3D7, available Laverania, P. relictum, and P. gallinaceum MSP2-like sequences on PlasmoDB and identified through a BLAST search on the NCBI server. P. sp. 1, 2, and 3 represent MSP2 sequences identified using a BLAST search that are indicated to be from an unspecific Laverania spp. parasite. Sequences presented are not aligned, with the exceptions of the three P. reichenowi and three unspecified Laverania sequences in order to highlight the conserved sequence despite an insertion. Insertions that are considered to break up the small amino acid and/or repeat structure for the purposes of this comparison are underlined in black. Repeats, both conserved and degenerate, are underlined in grey. Amino acids are coloured according to their properties (RasMol). (B) Amino acid sequence properties for the N-terminal repeat region of P. falciparum 3D7, other Laverania species (including three Laverania sequences of unknown speciation), P. relictum and P. gallinaceum MSP2. Truncated species names are: 5 (P. reichenowi XM_012905494.2), 12 (Plasmodium sp. 1 MAP-2013 JX899678), 13 (Plasmodium sp. 2 MAP-2013 JX893169), and 14 (Plasmodium sp. 2 MAP-2013 JX893170).
Figure 2
PfMSP2 is not essential for growth in vitro of Pf3D7 blood stage parasites.
(A–B) Schematic and agarose gel showing integration of knock-out construct (band A+C) in the msp2 gene locus and absence of the msp2 sequence (band A+B) for a representative gel for Pf3D7 ΔMSP2 Clone 1. (C) Western blot of late schizont protein extracts confirms no PfMSP2 is expressed in a representative blot of Pf3D7 ΔMSP2 Clone 1. PfMSP2 detected by anti-PfMSP2 2F2 3D7 mAb with PfAldolase (upper blot) or PfGAP45 (lower blot) serving as loading and stage of expression controls, respectively. Representative image is shown. (D) Distribution of key merozoite surface proteins for Pf3D7 wild-type (WT) compared to Pf3D7 ΔMSP2 Clone 1 parasites visualised by immunofluorescence. PfMSP2 (magenta), the nucleus stained by 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI) (cyan) and PfAMA1 (yellow, top two rows) or PfMSP1–19 (yellow, bottom two rows), and the coloured merge of the preceding panels. Scale bar = 0.7 µm. Representative images shown from a minimum of 10 schizonts imaged per condition. (E–F) Growth of Pf3D7 WT compared to Pf3D7 ΔMSP2 Clone 1 and 2 P. falciparum parasites, measured as fold increase in parasitaemia, over one (48 hr) or two (96 hr) cycles in either standard (still- (E)) or shaking (F) conditions, with no significant difference (p>0.05) between Pf3D7 ΔMSP2 Clone 1 and 2 parasite growth rates and WT control between standard or shaking conditions or one or two cycles. Parasitaemia was determined by flow cytometry at the start and end to calculate fold increase. Graph displays mean ± SD of three independent experiments performed with technical triplicates. Significance determined by unpaired t-test with p<0.05 deemed significant. Original PCR and western blot data available in Figure 2—source data 1 and 2.
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Figure 2—source data 1
PDF files containing original PCR gel and western blot images for Figure 2.
Relevant bands and treatments indicated.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig2-data1-v1.zip
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Figure 2—source data 2
Unannotated original files containing PCR gel and western blot images for Figure 2.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig2-data2-v1.zip
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Figure 2—source data 3
All the source data for the graphs (E,F) in Figure 2.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig2-data3-v1.xlsx
Figure 3
PfDd2 does not require MSP2 for asexual growth in vitro.
(A–B) Successful integration of knock-out (KO) construct (schematic in A) into the msp2 gene locus of PfDd2 ΔMSP2 was confirmed by PCR of genomic DNA (representative gel for Clone 1: primers A+B amplify wild-type [WT] locus, primers D+E amplify integrated KO construct). (C) Loss of PfMSP2 expression (representative blot for PfDd2 ΔMSP2 Clone 1) was demonstrated by western blot of schizont protein extract with PfMSP2 detected by anti-PfMSP2 FC27 and anti-PfEXP2 as loading control. Representative image is shown. (D) Growth of PfDd2 WT P. falciparum parasites and PfDd2 ΔMSP2 Clone 1 and 2 parasites over one (48 hr) or two (96 hr) cycles with no significant difference (p>0.05) between PfDd2 ΔMSP2 Clone 1 and 2 parasite growth rates and WT control for either one or two cycles. Parasitaemia was determined by flow cytometry at the start and end to calculate fold increase. Graph displays mean ± SD of three independent experiments performed with technical triplicates. (E–H) Key parameters of merozoite invasion were measured for both PfDd2 WT (n=43) and PfDd2 ΔMSP2 Clone 1 (n=35) parasites that had successfully invaded a red blood cell (RBC) using live-cell imaging of merozoite invasion. Time to merozoite attachment to RBCs (E), length (F), and strength (G) of RBC deformation, and time to complete merozoite invasion (H) was measured by live microscopy. Deformation scores are as defined by Weiss et al., 2015, with 1=weak deformation of the RBC membrane at the point of contact, 2=strong deformation leading to the RBC membrane extending up the sides of the merozoite and changes in RBC membrane curvature beyond the point of contact, and 3=extreme deformation indicated by the merozoite being deeply embedded in the RBC membrane and strong deformation of the RBC well beyond the point of contact. Significance determined by unpaired t-test with p<0.05 deemed significant. Original PCR and western blot data available in Figure 3—source data 1 and 2.
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Figure 3—source data 1
PDF files containing original PCR gel and western blot images for Figure 2.
Relevant bands and treatments indicated.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig3-data1-v1.zip
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Figure 3—source data 2
Unannotated original files containing PCR gel and western blot images for Figure 2.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig3-data2-v1.zip
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Figure 3—source data 3
All the source data for the graphs (D,E,FG,H) in Figure 3.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig3-data3-v1.xlsx
Figure 4 with 1 supplement
The impact of the loss of PfMSP2 on expression of known merozoite invasion genes and invasion pathway utilisation.
(A) Impact of PfMSP2 knock-out (KO) on schizont transcript abundance was assessed by qPCR for genes located in proximity to Pfmsp2 on chromosome 2. Changes in expression between Pf3D7 wild-type (WT) and Pf3D7 ΔMSP2 Clone 1 parasites were determined by qPCR relative to pfaldolase expression with pfsub1 serving as a schizont stage control. Graph displays mean ± SD of three independent RNA harvests with no significant difference (p>0.05) between MSP4 and MSP5 expression for Pf3D7 ΔMSP2 Clone 1 parasites identified. (B) Selective enzymatic cleavage of key RBC receptors showed no difference in invasion preference between Pf3D7 MSP2 WT and Pf3D7 ΔMSP2 Clone 1 parasites. Parasitaemia was determined by flow cytometry and compared to growth in non-treated control RBCs. Graph displays mean ± SD of three independent experiments with no significant difference (p>0.05) between enzyme-treated and non-enzyme-treated Pf3D7 ΔMSP2 Clone 1 parasite growth. (C) Log2(fold change) for differentially expressed genes, including multigene families, between the transcriptome of Pf3D7 WT and Pf3D7 ΔMSP2 Clone 1 schizonts. Plot represents the results of four independent schizont RNA harvests for Pf3D7 WT and Pf3D7 ΔMSP2 parasites, and red lines differentiate genes with a log2(fold change)>0.5 and <−0.5 with adjusted p-value<0.01. Genes shaded blue represent those genes that were found to have an average log2(fold change)>0.5 (dark blue) or <−0.5 (light blue) across the four replicate samples compared. Significance determined as below p<0.05 after correction for multiple testing.
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Figure 4—source data 1
All the source data for the graphs (A,B) in Figure 4.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Coverage plot (IGV) showing the absence of majority of msp2 in schizont transcriptome of Pf3D7 ΔMSP2 Clone 1 parasites but msp2 presence in Pf3D7 MSP2 wild-type (WT).
Small section of msp2 transcribed in Pf3D7 ΔMSP2 parasites corresponds to the end of the homology region and primarily covers the signal peptide.
Figure 5
Impact of PfMSP2 removal on efficacy of antibodies targeting other merozoite surface-exposed antigens.
Changes in antibody efficacy in the absence of PfMSP2 were assessed by measuring changes in antibody invasion inhibition and subsequent growth compared to growth in the absence of antibody for both P. falciparum 3D7 and Dd2 wild-type (WT), and 3D7 ΔMSP2 Clone 1 and Dd2 ΔMSP2 Clone 1, parasites over two cycles. (A) Rabbit (Rb) IgG raised against merozoite antigens of the Pf3D7 EBA/Rh family. (B) Rabbit sera raised against PfDd2 EBA175. (C) Rabbit IgG raised against Pf3D7 Rh5. (D) Nanobody (nAb) to Pf3D7 PTRAMP. (E–F) Nanobody and Fc-tagged nanobody to Pf3D7 CSS. (G) The invasion inhibitory glycosaminoglycan heparin. (H) Rabbit sera raised against Pf3D7 MSP1–19 (different vaccinated rabbit sera identified by numbers). Graph displays mean ± SD of three different experiments. Significance was determined by unpaired t-test when only a single concentration point was tested and for IC50 comparisons an extra Sum-of-Squares F test (best-fit LogIC50) was performed with p<0.05 deemed significant.
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Figure 5—source data 1
All the source data for the graphs (A,B,C,D,E,F,G,H) in Figure 5.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig5-data1-v1.xlsx
Figure 6
Absence of PfMSP2 from the merozoite surface impacts invasion inhibition by PfAMA1 antibodies.
Pf3D7 (A, D–F, H, I) and PfDd2 (B, C, G) express different PfMSP2 alleles and different PfAMA1 alleles, yet both showed altered anti-AMA1 antibody growth inhibition for 3D7 ΔMSP2 Clone 1 and Dd2 ΔMSP2 Clone 1 parasites compared to parental parasites. The effect was seen with serum (A–B; different vaccinated rabbit (Rb) sera identified by numbers), purified rabbit and mouse monoclonal (mAb) antibodies (C–E) and i-bodies (ibA) (F–I). Final parasitaemia was determined by flow cytometry and compared to control. Graph displays mean ± SD of three or four independent experiments. Significance was determined by unpaired t-test when only a single concentration point was tested and for IC50 comparisons an extra Sum-of-Squares F test (best-fit LogIC50) was performed with p<0.05 deemed significant.
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Figure 6—source data 1
All the source data for the graphs (A,B,C,D,E,F,G,H,I) in Figure 6.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig6-data1-v1.xlsx
Figure 7 with 1 supplement
Quantitative fluorescence microscopy to assess whether differential binding may explain the increased potency of anti-PfAMA1 invasion-inhibitory antibodies in the absence of PfMSP2.
Fluorescence intensity of fluorescently tagged anti-PfAMA1 i-body (ibA) WD34-mCherry (A) and WD33-eGFP (B) for both Pf3D7 wild-type (WT) and Pf3D7 ΔMSP2 Clone 1 parasites, with a representative image for i-body WD34-mCherry. Nucleus in blue, i-body signal in red. Scale bar = 4 µm. Two independent experiments were performed with significance determined by unpaired t-test with p<0.05 deemed significant. The lower overall mCherry signal required a higher antibody concentration (240 ng/mL) to have a comparable intensity measure to the eGFP-tagged antibody (120 ng/mL) for Pf3D7 WT merozoites. (C) Read-out of the surface plasmon resonance (SPR) antibody on-rate (association constant) for anti-PfAMA1 mAb 4G2 and i-body WD34-Fc (mouse Fc) binding to PfAMA1 in the presence or absence of PfMSP2 protein. Data represents the mean of three experiments with significance determined by unpaired t-test with p<0.05 deemed significant. (D) ELISA-based assessment of the anti-PfAMA1 mAb 4G2 and i-body WD34-Fc antibody binding levels to recombinant Pf3D7 AMA1 in the presence or absence of recombinant Pf3D7 MSP2. PBS control demonstrates background fluorescence. Dashed orange line provides a guide for peak absorbance levels. Anti-PfMSP2 mAb shows increasing concentrations of PfMSP2 protein results in decreased binding of mAb 4G2 and i-body WD34-Fc. Data represents the mean of three experiments and error bars are ± SD.
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Figure 7—source data 1
All the source data for the graphs (A,B,C,D) in Figure 7.
- https://cdn.elifesciences.org/articles/107603/elife-107603-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Enzyme-linked immunosorbent assay (ELISA) based assessment of the anti-PfAMA1 mAb 4G2 and i-body WD34-Fc antibody binding levels to recombinant Pf3D7 AMA1 in the presence or absence of: (A) the intrinsically disordered 40 kDa Pf3D7 MSP4 and (B) the structured immunoglobulin domain of the neural cell adhesion molecule (NCAM, CD56, 16 kDa).
PBS control demonstrates background fluorescence. Dashed orange line provides a guide for peak absorbance levels. Anti-His antibody detects the His-Tag of the recombinant PfMSP4 and anti-NCAM antibody detects the NCAM protein. Increasing concentrations of PfMSP4 and NCAM did not result in any noticeable change in binding of mAb 4G2 and i-body WD34-Fc. Data represents the mean of three (PfMSP4) and two (NCAM) experiments and error bars are ± SD.
Tables
Table 1
Genes significantly up or down-regulated with Pf3D7 MSP2 KO.
| Gene Name | log2-Fold Change | p-value | Name |
|---|---|---|---|
| PF3D7_0202400 | 2.547546545 | 1.09E-36 | translation-enhancing factor |
| PF3D7_1348000 | 1.604559487 | 0.000176 | conserved Plasmodium protein, unknown function |
| PF3D7_1423500 | 1.411878628 | 4.31E-08 | conserved Plasmodium protein, unknown function |
| PF3D7_1208200 | 1.384846624 | 3.59E-05 | cysteine repeat modular protein 3 |
| PF3D7_1461800 | 1.376899732 | 9.97E-05 | conserved Plasmodium protein, unknown function |
| PF3D7_0909100 | 1.155215486 | 4.07E-05 | conserved Plasmodium membrane protein, unknown function |
| PF3D7_1426500 | 1.021678659 | 1.52E-05 | ABC transporter G family member 2 |
| PF3D7_0322200 | 1.014455112 | 0.000313 | conserved protein, unknown function |
| PF3D7_1372100 | –2.436194076 | 9.31E-18 | Plasmodium exported protein (PHISTb), unknown function |
| PF3D7_1149400 | –2.748153616 | 1.72E-25 | Plasmodium exported protein, unknown function |
| PF3D7_0206800 | –4.324734054 | 9.44E-13 | merozoite surface protein 2 |
Additional files
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Supplementary file 1
Primers used to generate MSP2 knock-out (KO) lines.
- https://cdn.elifesciences.org/articles/107603/elife-107603-supp1-v1.pdf
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Supplementary file 2
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/107603/elife-107603-supp2-v1.pdf
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Supplementary file 3
Enzymes used to remove/inactivate red blood cell (RBC) receptors and target residues of the enzyme.
- https://cdn.elifesciences.org/articles/107603/elife-107603-supp3-v1.pdf
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Supplementary file 4
Primers used for RT-qPCR of P. falciparum MSP2, MSP5, MSP4, and controls.
- https://cdn.elifesciences.org/articles/107603/elife-107603-supp4-v1.pdf
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Supplementary file 5
Differential gene expression between wild-type (WT) and MSP2 knock-out (KO) parasites.
- https://cdn.elifesciences.org/articles/107603/elife-107603-supp5-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107603/elife-107603-mdarchecklist1-v1.pdf
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An abundant merozoite surface protein of Plasmodium falciparum modulates susceptibility to inhibitory antibodies
eLife 14:RP107603.
https://doi.org/10.7554/eLife.107603.4
