Journal: Malaria Journal

Article Title: Genetic diversity of three surface protein genes in Plasmodium malariae from three Asian countries

doi: 10.1186/s12936-018-2176-x

Figure Lengend Snippet: Sliding window plots of the average pair-wise nucleotide diversity (π). The nucleotide diversity of six Plasmodium species trap (red), ama1 (green) and p48/45 (blue) were plotted by a sliding window with a window size of 60 bp, a step size of 3 bp, and excluding sites with gaps

Article Snippet: Population genetics of P. malariae was analysed based on trap , ama1 , and p48/45 gene polymorphism.

Techniques:

Journal: Malaria Journal

Article Title: Genetic diversity of three surface protein genes in Plasmodium malariae from three Asian countries

doi: 10.1186/s12936-018-2176-x

Figure Lengend Snippet: Average nucleotide diversity and haplotype diversity of three surface genes of P. malariae , P. falciparum , and P. vivax

Article Snippet: Population genetics of P. malariae was analysed based on trap , ama1 , and p48/45 gene polymorphism.

Techniques:

Journal: Malaria Journal

Article Title: Genetic diversity of three surface protein genes in Plasmodium malariae from three Asian countries

doi: 10.1186/s12936-018-2176-x

Figure Lengend Snippet: Schematic structure and amino-acid variants of P. malariae AMA1. P. malariae AMA1 contains apical membrane antigen 1 domain. The alignment of 4 reference sequences and 43 PmAMA1 isolates from this study revealed 20 variant types, in which the variant 5 (GYKKNDKPLKVIN) is the highest frequency found in this study

Article Snippet: Population genetics of P. malariae was analysed based on trap , ama1 , and p48/45 gene polymorphism.

Techniques: Membrane, Variant Assay

Journal: PLoS Pathogens

Article Title: The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites

doi: 10.1371/journal.ppat.1001276

Figure Lengend Snippet: (A) Co-immunoprecipitation of TgAMA1 and TgRON2 after 0.6% SDS lysis and Triton X100 renaturation of tachyzoite extract: anti-RON2-1 allows the recovery of both proteins, while anti-RON2-2 only recovers TgRON2. (B) Transiently transfected BHK-21 cells express TgAMA1 at their surface in its native conformation, with the ectodomain exposed outside (revealed with antibody B3.90, “ -ecto”) and the cytosolic tail only detected after permeabilization (revealed with antibody CL22, “ -tail”). (C) TgAMA1-expressing BHK-21 cells were incubated with 0.1 µg/ml TgRON2-2 (a GST-fusion protein), washed and the binding of recombinant TgRON2 fragment to TgAMA1-expressing cells was revealed with anti-GST antibody. Untransfected BHK-21 cell on the left showed no TgRON2 binding. (D) Far Western blot analysis of TgRON2-2 binding to native TgAMA1. Extracts of tachyzoites expressing wild-type levels of AMA1 (WT) or depleted for this protein (KOiAMA1+Atc) were separated by SDS-PAGE and transferred on a nitrocellulose membrane, which was incubated with GST alone or TgRON2-2 and revealed with anti-GST. Incubation with TgRON2-2 revealed a band corresponding to the size of TgAMA1, as shown by specific antibodies and absent from the AMA1-depleted cell line extracts, while anti-GST antibody did not react with tachyzoites. Anti-SAG1 was used as a loading control. (E) TgRON2-2 binds specifically to surface-exposed TgAMA1. Fixed extracellular conditional KOi AMA1 tachyzoites induced (+Atc) to repress AMA1 expression, or non-induced (no Atc), were incubated with TgRON2-2, which was then revealed with anti-GST antibody. TgAMA1 was revealed with anti-ectodomain B3.90 antibody. (F) TgRON2-2 binds specifically to TgAMA1 in micronemes. Fixed intracellular induced or non-induced conditional KOi AMA1 tachyzoites were permeabilized and incubated with TgRON2-2, which labelled micronemes only when TgAMA1 was expressed. MIC3, an unrelated microneme marker, was used as a control.

Article Snippet: Generation of a complete synthetic variant gene of Pf3D7 AMA1 utilizing mouse codon usage and lacking putative N-glycosylation sites as previously described was performed by Genscript, USA.

Techniques: Immunoprecipitation, Lysis, Transfection, Expressing, Incubation, Binding Assay, Recombinant, Far Western Blot, SDS Page, Membrane, Control, Marker

Journal: PLoS Pathogens

Article Title: The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites

doi: 10.1371/journal.ppat.1001276

Figure Lengend Snippet: (A) Extracellular tachyzoites were pre-incubated with either GST or TgRON2-2 at 125 µg/ml (3.75 µM) or 250 µg/ml (7.5 µM), before being added to confluent HFF monolayers for 30 min. Significant decrease in T. gondii invasion ( t -test, ** p<0.01) was observed in the presence of TgRON2-2 at a concentration of 250 µg/ml. (B) Decreasing TgAMA1 expression at the surface is correlated with an enhanced effect of TgRON2-2 on invasion. RH hxgprt- and KOi AMA1 strains were compared for their invasion efficiency in the presence of GST or TgRON2-2 at 200 µg/ml (6 µM). The invasion process was stopped by cell fixation after 30 min or 5 min. In both cases, KOi AMA1 shows a more pronounced invasion defect in presence of TgRON2-2 compared to RH hxgprt- ( t -test, * p<0.05). Values represent means ± standard deviations (SD), n = 3, from a representative experiment out of 4 independent assays. (C) Western blot analysis of TgAMA1 expression levels in RH strain (1) or in the KOi AMA1 strain grown in absence of Atc (2). Anti-ROP1 antibody was used as a loading control.

Article Snippet: Generation of a complete synthetic variant gene of Pf3D7 AMA1 utilizing mouse codon usage and lacking putative N-glycosylation sites as previously described was performed by Genscript, USA.

Techniques: Incubation, Concentration Assay, Expressing, Western Blot, Control

Journal: PLoS Pathogens

Article Title: The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites

doi: 10.1371/journal.ppat.1001276

Figure Lengend Snippet: (A) Pf3D7-AMA1 displays a type I TM topology at the plasma membrane of BHK-21 cells. PfAMA1 transfected BHK-21 cells were subjected to IFA. Mouse MAb F8.12.19 recognizes extracellular PfAMA1 domain III that is accessible with or without permeabilization, whereas anti-Myc antibody labels the intracellular C-terminal Myc tag in permeabilized cells only. (B) PfAMA1 transfected BHK-21 cells were incubated with 20 µg/ml of GST or PfRON2-5. Recombinant proteins were detected using anti-GST antibody.

Article Snippet: Generation of a complete synthetic variant gene of Pf3D7 AMA1 utilizing mouse codon usage and lacking putative N-glycosylation sites as previously described was performed by Genscript, USA.

Techniques: Clinical Proteomics, Membrane, Transfection, Incubation, Recombinant

Journal: PLoS Pathogens

Article Title: The RON2-AMA1 Interaction is a Critical Step in Moving Junction-Dependent Invasion by Apicomplexan Parasites

doi: 10.1371/journal.ppat.1001276

Figure Lengend Snippet: ELISAs were carried out with increasing concentrations of Pf3D7-RON2-5 (A) or TgRON2-2 (B) and recombinant AMA1 proteins from Pf3D7, PvSalI, TgRH, or PfFVO (C) strains coated at 1 µg/ml. GST alone was used as a control. (D) Competitive ELISAs were performed using 20 µg/ml Pf3D7-RON2-5, 1 µg/ml Pf3D7-AMA1 and increasing concentrations of R1 peptide, or P8 control peptide. Values represent means ± SD, n = 2, from a representative experiment out of 4 independent assays.

Article Snippet: Generation of a complete synthetic variant gene of Pf3D7 AMA1 utilizing mouse codon usage and lacking putative N-glycosylation sites as previously described was performed by Genscript, USA.

Techniques: Recombinant, Control

Journal: PLoS ONE

Article Title: Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

doi: 10.1371/journal.pone.0006559

Figure Lengend Snippet: Immune responses of vaccine groups prior to challenge.

Article Snippet: Synthetic peptides for the CSP and AMA1 antigens were produced by Pepscan (Lelystad, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Sterile Protection against Plasmodium knowlesi in Rhesus Monkeys from a Malaria Vaccine: Comparison of Heterologous Prime Boost Strategies

doi: 10.1371/journal.pone.0006559

Figure Lengend Snippet: Cox Proportional Hazards Analysis of immune responses and parasitemia.

Article Snippet: Synthetic peptides for the CSP and AMA1 antigens were produced by Pepscan (Lelystad, The Netherlands).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Sequence analysis of BdAMA1 and NcAMA1 compared with AMA1 proteins for which structures have been determined. Sequences are numbered from the initiation methionine in the signal sequence. Top secondary structure elements refer to TgAMA1, bottom elements refer to PvAMA1. Cyan arrows indicate DI limits; blue—DII, purple—DIII. Orange numbers indicate disulfide connectivity, with top DIII numbers referring to Tg/NcAMA1 and bottom DIII numbers referring to Pf/PvAMA1. The highly variable DII loop is indicated by a blue box. Bottom—phylogenetic tree of the five structurally characterized AMA1 proteins generated in MEGA4 with 500 bootstrap replicates from a MUSCLE alignment of the fully processed ectodomains. Domain boundaries of the fully processed Bd/NcAMA1 ectodomains were defined based on the paradigm established for previously characterized AMA1s.37, 43, 45, 46, 56 BdAMA1 DI spans residues from Pro84 to Pro305 (numbering based on the initiation methionine in the signal sequence), DII from Met307 to Pro445 and DIII from Phe446 to Lys523, while NcAMA1 DI spans residues from Thr62 to Pro281, DII from Asn282 to Ile410 and DIII from Ile411 to Ala481.

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques: Sequencing, Generated

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Extreme divergence in DIII of AMA1.(A) Top—solid surfaces of NcAMA1, BdAMA1, and PvAMA1 (PDB ID: 1W8K) DI (cyan) and DII (blue), with secondary structure of DIII (purple). Disulphide bonds in DIII shown as yellow sticks. Bottom—corresponding DIII topology diagrams with cysteines depicted as yellow stars, disulfide bonds as yellow dotted lines, and disordered regions of DIII as purple dotted lines. (B) Phylogenetic analysis in MEGA4 with 500 bootstrap replicates based off of MUSCLE alignments of DIII domains (as indicated by purple arrows in Fig. 1) supports divergence of the DIII domains and distant clustering of BdAMA1 DIII with Nc/TgAMA1 DIII.

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Cross-genera reactivity in the AMA1–RON2 interaction.(A) Apical surface views of BdAMA1 (left) and NcAMA1 (right) with the DII loop removed to the point of corresponding disorder in the TgAMA1-TgRON2sp co-structure. DI/DII are shown as semi-transparent cyan/blue surfaces, with secondary structure of loops visible, and un-modeled loops indicated by dotted lines. Black bars indicate the apical groove and black numbers represent the apical surface loops as defined by previous AMA1 structures. (B) Native gel electrophoresis assays reveal a level of structural plasticity in the AMA1–RON2 interaction. BdAMA1 does not interact with TgRON2sp (left), but NcAMA1 is able to bind TgRON2sp (middle) at the same molar ratio as TgAMA1 (right). (C) Fluorescence polarization assay with recombinant TgAMA1, NcAMA1, and BdAMA1, and 5FAM-TgRON2sp. Error bars indicate SEM for triplicate samples. EC50 values calculated from a sigmoidal nonlinear regression model in GraphPad Prism 5.0. mP—millipolarization units; ND—not determined.

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques: Nucleic Acid Electrophoresis, Fluorescence, Recombinant

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Structural plasticity and specificity of the AMA1–RON2 interaction. (A) Sequence alignment of RON2 sequences for the regions that align with TgRON2sp, with boxes around the conserved RON2 Pro, the single difference between TgRON2 and NcRON2 in this region, and the cystine loop. (B) TgAMA1 apical surface (gray) with TgRON2sp secondary structure (green) (PDB ID: 2Y8T) and residues of interest shown as sticks; black numbers indicate regions of conservation and divergence with Bd/NcAMA1 under investigation. 1—Top: overlay of TgRON2sp onto BdAMA1 shows that the AMA1 Tyr trio that accommodates a key RON2 Pro (green sticks) is substituted in BdAMA1 with a Tyr/Tyr/His (cyan sticks) that conserves the architecture of the pocket. Bottom: overlay of TgRON2sp (green) onto NcAMA1 shows that the complete Tyr trio is conserved in NcAMA1 (cyan sticks). Gray mesh: 2Fo-Fc electron density maps around the pocket defining residues contoured at 1.0 σ. 2—The central TgAMA1 Tyr230 (gray) forms a functionally important bifurcated hydrogen bond with the backbone of TgRON2 Val1311 (green). Plasticity may allow similar binding between NcAMA1 Ser224 (cyan) and the corresponding NcRON2 Ala1310. (C) Left—End-on view of PfRON2sp1 (orange secondary structure) bound to PfAMA1 (gray surface) (PDB ID: 3ZWZ) shows a deep pocket on the surface of AMA1 into which a critical RON2 arginine residue anchors (orange sticks, dotted box). Right—An overlay of PfRON2sp1 on BdAMA1 (gray surface) shows that a similar pocket is not present (dotted box). (D) Electrostatic surface, end-on views of TgAMA1 bound to TgRON2sp (green secondary structure with cystine loop tip side chains shown as sticks) (left; PDB ID: 2Y8T), and NcAMA1 (middle) and BdAMA1 (right) overlayed on the TgAMA1-TgRON2sp co-structure, reveal key differences in the chemical nature of the cystine loop binding region. An interactive view is available in the electronic version of the article.

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques: Sequencing, Binding Assay, Residue

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Identification of surface pockets ideal for targeting with therapeutics. (A) Left—apical view of an overlay of three structurally characterized ligands (shown as secondary structure, with residues occupying pockets of interest shown as sticks)—TgRON2sp (green; PDB ID: 2Y8T), PfRON2sp1 (orange; PDB ID: 3ZWZ), R1_major (yellow; PDB ID: 3SRJ)—on the surface of TgAMA1 (gray). Inset—enlarged apical view for the identification of three surface pockets with structural and chemical features conserved across all the structurally characterized AMA1 proteins. Right—schematic representation showing the relative positions of the three pockets in a triangular organization, with Pocket 2 in a higher plane than Pockets 1 and 3. Pocket 2 is equidistant from 1 and 3 (11 Å), while Pockets 1 and 3 are separated by roughly 13 Å. (B) Left—apical surface of TgAMA1 (gray) in the same orientation as (A), with the residues defining Pocket 1 shown purple; 2, light blue; 3, bright blue. Right—end-on view highlighting the depth of Pocket 3 and the backstop nature of Pocket 2.

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Babesia divergens and Neospora caninum apical membrane antigen 1 structures reveal selectivity and plasticity in apicomplexan parasite host cell invasion

doi: 10.1002/pro.2193

Figure Lengend Snippet: Residues Lining the Conserved Apical Surface Pockets of AMA1

Article Snippet: Sequences encoding the fully processed ectoplasmic domains of Bd AMA1 and Nc AMA1 were synthesized by GenScript, codon optimized for insect cells, and subcloned into a modified pAcGP67b vector (Pharmingen) incorporating a C-terminal hexahistidine tag separated from ama1 by a thrombin cleavage site.

Techniques:

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Inhibitory activity of monoclonal antibodies against Pf AMA1 following mutation of Loop1E. A 3D structure of AMA1 with arrows indicating the approximate binding sites of mAbs used in this study (1B10, 4E8, 4E11, 1E10, and 5A6). Other known inhibitory mAbs 1F9 and 4G2 are shown for reference. B Western blots of schizont material from P. falciparum expressing wild-type Pf AMA1 (W2-3D7) or Pv AMA1 with Pv Loop1E (W2-3D7 Pv Loop1E) were probed with the Loop1E binding mAbs 4E11, 4E8, 1B10 or the domain 2 binding mAb 5A6 as a loading control. All antibodies were tested at a final concentration of 2.5 µg/mL IgG. C Differential growth inhibition of transgenic W2-3D7 and W2-3D7 Pv Loop1E parasite lines by mAbs 4E11, 4E8, 1B10, 5A6, and 1E10 or by Pf RON2 peptide. The Y -axis shows % inhibition; mean and standard deviation of two inhibition assays performed in triplicate wells. Statistical analysis was by paired t test; *indicates p < 0.05

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Activity Assay, Mutagenesis, Binding Assay, Western Blot, Expressing, Concentration Assay, Inhibition, Transgenic Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Similarities and differences in RON2 and AMA1 amino acid sequences between Plasmodium spp. Neighbour-joining dendrograms (left) showing similarities between the amino acid sequences of Plasmodium spp. For RON2L ( A ) and the AMA1 ectodomain ( B ). In both cases, species group as expected by subgenera; the rodent Vinckeia species group together, as do species of the Plasmodium subgenus (including P. vivax and P. knowlesi ), and the Laverania subgenus ( P. falciparum and P. reichenowi ). The distance matrices (right) also support this grouping. Notably, sequences from P. vivax and P. knowlesi are separated by a very low distance, with 0.10 and 0.15 for RON2L and the AMA1 ectodomain, respectively

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques:

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Conserved and divergent functions of RON2-loop among P. falciparum, P. vivax, and P. knowlesi . A Alignment of the RON2L peptide sequences from P. falciparum (Pf), P. vivax (Pv) and P. knowlesi (Pk) orthologues. Amino acids conserved between Pv RON2 and Pk RON2 are in red. Amino acids that form the disulfide-bonded β-hairpin loop are shaded in orange B Invasion inhibition of transgenic P. falciparum expressing Pf AMA1 (W2-3D7) or Pv AMA1 (W2- Pv AMA1) or P. knowlesi (strain H) by Pf RON2, Pv RON2, Pk RON2 and the control Pf RON3 peptides. C Binding of Pv RON2L peptide to recombinant Pf AMA1, Pv AMA1, Pk AMA1 or Pfs230 (negative control). Data from 2 or more experiments in duplicate and show mean ± SD

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Inhibition, Transgenic Assay, Expressing, Binding Assay, Recombinant, Negative Control

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: The central β-hairpin loop of RON2L peptides mediate species-specific inhibition of invasion. A Alignment of P. falciparum ( Pf ) RON2L and the chimeric P. vivax flank/ P. falciparum central β-hairpin loop hybrid RON2L ( PvPf RON2L) peptides. Amino acids that differ between Pf RON2L and Pv RON2L are in red. Amino acids that form the β-hairpin loop are shaded in orange. B Invasion inhibition of P. falciparum expressing Pf AMA1 (W2-3D7) or Pv AMA1 (W2- Pv AMA1) by 200 μg/mL Pf RON2L peptides. C Inhibition of transgenic P. falciparum expressing Pf AMA1 (W2-3D7) or Pv AMA1 (W2- Pv AMA1) by 200 μg/mL PvPf RON2L. D Inhibition of transgenic P. falciparum expressing Pf AMA1 (W2-3D7) by Pf RON2L and PvPf RON2L peptides. E Alignment of Pv RON2L ( Pv RON2L) and the chimeric P. falciparum flank/ P. vivax central β-hairpin loop hybrid RON2L ( PfPv RON2L) peptides. Conserved amino acids are in red. Amino acids that form the β-hairpin loop are shaded in orange. F Inhibition of transgenic P. falciparum expressing Pf AMA1 (W2-3D7) or Pv AMA1 (W2- Pv AMA1) by 200 μg/mL PfPv RON2L

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Differences between Plasmodium species in AMA1 amino acids that interact with RON2-loop, and generation of a hybrid Pv AMA1- Pf Loop1E mutant parasite line. A Key P. falciparum ( Pf ) AMA1 amino acid residues that have been identified to play a role in binding to Pf RON2 aligned against those found at the same relative position in Pv AMA1 and Pk AMA1 (residue numbers are Pf AMA1 specific). Amino acids conserved across all three species are in red; except amino acid 232, which is the same in Pf AMA1 and Pv AMA1, but differs in Pk AMA1. Amino acids conserved between Pv AMA1 and Pk AMA1 only are in green. Amino acids located in the Loop1E domain (222–234) are shaded in orange. B Alignment of Pf AMA1 and Pv AMA1 Loop1E amino acid sequences. Proposed Pf RON2 interacting residues are in red. Pv AMA1 (Palo Alto) residues that differ from Pf AMA1 (3D7) are shown in magenta and an alternative amino acid present in other Pv AMA1 alleles is shown in orange. The hybrid Pv AMA1 incorporating Pf AMA1 Loop1E is shown, with Pf AMA1-specific amino acid residues in blue. C Transgenic P. falciparum expressing a chimeric Pv AMA1 with the Pf AMA1-Loop1E sequence (denoted Pv AMA1 Pf Loop1E) was generated (see Supplementary Material, Figure S5). Analysis of Pv AMA1 Pf Loop1E protein expression was investigated by western blot. Schizont protein extracts were prepared from transgenic P. falciparum lines expressing either Pf AMA1 (W2-3D7), Pv AMA1 (W2- Pv AMA1) or Pv AMA1 Pf Loop1E (W2- Pv AMA1 Pf Loop1E) and run in non-reducing conditions (3–8% acrylamide gradient gel). Western blots were probed with anti-3D7 Pf AMA1 (3D7 allele) or anti- Pv AMA1 polyclonal antibodies (20 μg/mL IgG) raised in rabbits. Antibodies labelled the expected ~ 83 kDa and ~ 66 kDa species of Pf AMA1 protein, whereas a smaller ~ 81 kDa band is seen in transgenic parasites expressing Pv AMA1 and Pv AMA1 Pf Loop1E mutant proteins. Equal loading of schizont samples was confirmed by probing blots with anti- Pf RON4.2 mAb (5 μg/mL IgG)

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Mutagenesis, Binding Assay, Transgenic Assay, Expressing, Sequencing, Generated, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Parasites expressing Pv AMA1 incorporating Pf AMA1-Loop1E amino acids invade erythrocytes normally but display reduced inhibition by Pv RON2 peptide. A Merozoite invasion kinetics of transgenic P. falciparum ( Pf ) lines expressing P. vivax ( Pv ) AMA1 (W2- Pv AMA1) or Pv AMA1 Pf Loop1E (W2- Pv AMA1 Pf Loop1E) as measured by live-cell imaging at six consecutive stages of erythrocyte invasion. N = number of events filmed per stage of invasion. Statistical analysis by unpaired t test (invasions per schizont rupture) or Mann–Whitney U test (all other panels). The bar graphs are box-and-whisker type, showing the median (line), inter-quartile range (box) and range (whiskers). B Differential inhibition of transgenic W2- Pv AMA1 and W2- Pv AMA1 Pf Loop1E parasite lines by titrated Pf RON2, Pv RON2, Pk RON2 or the negative control Pf RON3 peptide. Mean and standard deviation of two assays performed in duplicate wells. Statistical analysis by paired t test for each individual peptide concentration. P -value score for all peptide concentrations ranges defined by the lower bar

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Expressing, Inhibition, Transgenic Assay, Live Cell Imaging, MANN-WHITNEY, Whisker Assay, Negative Control, Standard Deviation, Concentration Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Parasites expressing Pf AMA1 incorporating Pv AMA1-Loop1E amino acids replicate in erythrocytes normally but display reduced invasion inhibition by Pf RON2 peptide. A Alignment of Pf AMA1 and Pv AMA1 Loop1E amino acid sequences. Pf RON2 interacting residues are in red. Pv AMA1 (3D7 strain) residues that differ from Pf AMA1 are in magenta, and an alternative amino acid used by other Pv AMA1 alleles is in orange. The hybrid Pf AMA1 incorporating Pv AMA1-Loop1E is shown, with Pv AMA1-specific amino acid residues in blue. B Western blots of schizont material expressing Pf AMA1 (W2-3D7) or Pf AMA1 with Pv Loop1E (W2-3D7 Pv Loop1E) probed with 2.5 μg/mL 1F9 mAb, 20 μg/mL polyclonal rabbit anti-3D7 AMA1 IgG or 1:500 diluted polyclonal rabbit anti-HSP70 antisera. C P. falciparum multiplication rates were measured for W2-3D7 and W2-3D7 Pv Loop1E transgenic P. falciparum . Rates were calculated for parasite replication in static erythrocyte culture and suspended erythrocyte culture conditions. Mean and standard deviation of each condition was tested over five wells. Statistical analysis was done by paired t test. D, E Differential inhibition of parasites expressing Pf AMA1 and Pf AMA1- Pv Loop1E by 200 μg/mL Pf RON2 peptide ( D ) or 200 μg/mL Pv RON2 peptide ( E ). Mean and standard deviation of two assays was performed in triplicate wells. Statistical analysis was by paired t test

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Expressing, Inhibition, Western Blot, Transgenic Assay, Standard Deviation

Journal: Cellular and Molecular Life Sciences

Article Title: Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines

doi: 10.1007/s00018-023-04712-z

Figure Lengend Snippet: Models of possible AMA1 binding interactions for invasion. AMA1 is present on the merozoite surface and the RON2-loop (labelled as RON2L) is present on the erythrocyte surface. AMA1 is known to bind to the RON2-loop and new data suggest that there is an additional binding interaction of AMA1. In one model (panels A–C ), AMA1 binds to Receptor B, which is a proposed molecule on the erythrocyte surface, yet to be identified, that acts a second receptor for AMA1 binding. In the alternative model (panels D–E ), the second binding interaction of AMA1 is to a second site on RON2 that is separate to the RON2-loop. A, D . In P. falciparum parasites, Pf AMA1 binds to the Pf RON2-loop ( Pf RON2L) and either Receptor B ( A ) or another region of Pf RON2 ( D ) to mediate invasion. Key amino acids of Pf AMA1 Loop1E for binding to RON2 are indicated (G, N, M, and I), as identified in this study. B, E . In genetically modified P. falciparum parasites expressing Pv AMA1, Pv AMA1 does not bind to the Pf RON2-loop, but binds to either Receptor B ( B ) or another region of Pf RON2 ( E ), allowing invasion to occur. The lack of binding of Pv AMA1 to the Pf RON2-loop is due to differences in key amino acids of AMA1 Loop1E between P. vivax and P. falciparum (ASFV for Pv AMA1 and GNMI for Pf AMA1). C, F . In P. vivax and P. knowlesi parasites, Pv AMA1 and Pk AMA1 can bind to the Pv RON2-loop or Pk RON2-loop, as well as Receptor B ( C ) or another region on Pv RON2 or Pk RON2 ( F ). There is conservation of AMA1-RON2-loop binding between the two species. The amino acid sequences of the β-hairpin loop of RON2 are identical between P. vivax and P. knowlesi . Of the key RON2-interacting amino acids of AMA1 Loop1E, only one amino acid differs between P. vivax and P. knowlesi . The native conformation and surface orientation of RON2 is not currently fully defined

Article Snippet: Genes encoding Pf AMA1 (3D7 allele) and Pv AMA1 (Palo Alto allele) with mutated RON2-binding residues (AMA1-M) were generated by Spliced by Overlapping End (SOE) PCR, using a previously described codon-optimised Pf AMA1 and Pv AMA1 DNA as templates and synthetic oligonucleotides incorporating relevant DNA mutations (Geneworks Australia) [ , , ].

Techniques: Binding Assay, Genetically Modified, Expressing