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

Journal: eLife

Article Title: Group II truncated haemoglobin YjbI prevents reactive oxygen species-induced protein aggregation in Bacillus subtilis

doi: 10.7554/eLife.70467

Figure Lengend Snippet:

Article Snippet: * 1 NBRP: National BioResource Project, Japan. * 2 DNA fragments were synthesised with codon optimisation by GenScript. Bold letters indicate restriction enzyme sites. Initiation and termination codons are underlined. , , .

Techniques: Plasmid Preparation, Gene Expression, Over Expression, Sequencing