Figures and data
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 proteins 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 to 50) and orange very low confidence (<50). the Modelled structure of the N-terminal helical region is provided as an inset for P. falciparum 3D7 MSP2 in isolation, and also in alignment with the helical domain for P. falciparum 3D7 (green for all) for the other examples of Laverania and avian MSPs (other colours). (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.
Amino acids sequence properties for the N and C-terminal conserved, and N-terminal repeat, regions for available P. falciparum 3D7, Laverania (including 3 Laverania sequences of unknown speciation) P. relictum and P. gallinaceum MSP2 sequences.
PfMSP2 is not essential for growth in vitro of Pf3D7 blood stage parasites.
(A-B) Schematic and agarose gel showing integration of knockout construct (band A+C) in the msp2 gene locus and absence of the msp2 sequence from the Pf3D7 ΔMSP2 line (band A+B). Western Blot of late schizont protein extracts confirms no PfMSP2 is expressed in the Pf3D7 ΔMSP2 line. PfMSP2 detected by anti-PfMSP2 2F2 3D7 mAb with PfAldolase serving as loading control. Representative image shown. (D) Distribution of key merozoite surface proteins in the presence or absence of PfMSP2 was visualised by immunofluorescence. PfMSP2 (red for top two rows, purple in bottom two rows), the nucleus stained by DAPI (blue in merge) and PfAMA1 (purple, top two rows) or PfMSP1 19 (red, bottom two rows), and the coloured merge of the proceeding 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 P. falciparum parasites, measured as fold increase in parasitaemia, over one (48 hrs) or two (96 hrs) cycles in either standard (still- (E)) or shaking (F) conditions. Parasitaemia was determined by flow cytometry at the start and end to calculate fold increase. Graph displays mean ± S.D. of three independent experiments performed with technical triplicates. Significance determined by unpaired t-test with p< 0.05 deemed significant.
PfDd2 does not require MSP2 for asexual growth in vitro.
(A-B) Successful integration of KO construct (schematic in A) into the msp2 gene locus of PfDd2 ΔMSP2 was confirmed by PCR of genomic DNA (primers A+B amplify WT locus, primers D+E amplify integrated KO construct). (C) Loss of PfMSP2 expression was demonstrated by western blot of schizont protein extract with PfMSP2 detected by anti-PfMSP2 FC27 and anti-PfEXP2 as loading control. Representative image shown. (D) Growth of PfDd2 WT P. falciparum parasites and PfDd2 ΔMSP2 over one (48 hrs) or two (96 hrs) cycles. Parasitaemia was determined by flow cytometry at the start and end to calculate fold increase. Graph displays mean ± S.D. of three independent experiments performed with technical triplicates. (E-H) Key parameters of merozoite invasion were measured for both PfDd2 WT and PfDd2 ΔMSP2 parasites that had successfully invaded a 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) were measured by live microscopy. A minimum of 30 invading merozoites for each line were captured and assessed. Significance determined by unpaired t-test with p< 0.05 deemed significant.
The impact of the loss of PfMSP2 on expression of known merozoite invasion genes and invasion pathway utilization.
(A) Impact of PfMSP2 KO on transcription during schizonts was assessed by qPCR for genes located in proximity to Pfmsp2 on chromosome 2. Changes in expression between Pf3D7 WT and Pf3D7 ΔMSP2 parasites was determined by qPCR relative to pfaldolase expression with pfsub1 serving as a schizont stage control. Graph displays mean ± S.D. of three independent RNA harvests. (B) Selective enzymatic cleavage of key RBC receptors showed no difference in invasion preference between Pf3D7MSP2 WT and Pf3D7 ΔMSP2 parasites. Parasitaemia was determined by flow cytometry and compared to growth in non-treated control RBCs. Graph displays mean ± S.D. of three independent experiments. (C) Log2(fold change) for differentially expressed genes, including multigene families, between the transcriptome of Pf3D7 WT and Pf3D7 ΔMSP2 schizonts. Black lines differentiate genes with a log2 (fold change) > 0.5 and < −0.5 with adjusted p-value < 0.05. Four independent schizont RNA harvests were performed and analysed. Significance determined by t-test with p< 0.05 deemed significant.
Genes significantly up or down-regulated with Pf3D7 MSP2 KO
Impact of PfMSP2 removal on efficacy of antibodies targeting other merozoite surface-exposed antigens.
Changes in antibody efficacy in the absence of PfMSP2 was assessed by measuring changes in antibody invasion inhibition and subsequent growth compared to growth in the absence of antibody for both P. falciparum WT and ΔMSP2 parasites over 2 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 ± S.D. 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.
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 in the absence of PfMSP2. 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-J). Final parasitaemia was determined by flow cytometry and compared to control. Graph displays mean ± S.D. 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.
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 WT and Pf3D7 ΔMSP2, 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 binding to PfAMA1 in the presence or absence of PfMSP2 protein. Data represents the mean of 3 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 3 experiments and error bars are ±S.D.
Alignment of full-length amino acid protein sequences for all PfMSP2 like sequences on PlasmoDB.
Alignment done using MUSCLE alignment with default settings.
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).
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 3 P. reichenowi and 3 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. Respeats, both conserved and degenerate, are underlined in grey. Amino acids are coloured according to their properties (RasMol).
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).
Coverage plot (IGV) showing absence of majority of msp2 in schizont transcriptome of Pf3D7 DMSP2 parasites but msp2 presence in Pf3D7 MSP2WT.
Small section of msp2 transcribe in Pf3D7 ΔMSP2 parasites corresponds with the end of the homology region and primarily covers the signal peptide.
Primers used to generate MSP2 KO lines.
Antibodies used in this study.
Enzymes used to remove/inactivate RBC receptors and target residues of the enzyme.
