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). 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 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.

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). (C) 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 (upper blot) or PfGAP45 (lower blot) serving as loading and stage of expression controls respectively. Representative image shown. (D) Distribution of key merozoite surface proteins in the presence or absence of PfMSP2 was visualised by immunofluorescence. PfMSP2 (magenta), the nucleus stained by 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 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, with no measurable difference between parasite growth rates seen between standard or shaking 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. 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. 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 schizont transcript abundance 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. Plot represents the results for one 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.

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 s(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 (mouse 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.