Sequential roles for red blood cell binding proteins enable phased commitment to invasion for malaria parasites

Invasion of red blood cells (RBCs) by Plasmodium merozoites is critical to their continued survival within the host. Two major protein families, the Duffy binding-like proteins (DBPs/EBAs) and the reticulocyte binding like proteins (RBLs/RHs) have been studied extensively in P. falciparum and are hypothesized to have overlapping, but critical roles just prior to host cell entry. The zoonotic malaria parasite, P. knowlesi, has larger invasive merozoites and contains a smaller, less redundant, DBP and RBL repertoire than P. falciparum. One DBP (DBPα) and one RBL, normocyte binding protein Xa (NBPXa) are essential for invasion of human RBCs. Taking advantage of the unique biological features of P. knowlesi and iterative CRISPR-Cas9 genome editing, we determine the precise order of key invasion milestones and demonstrate distinct roles for each family. These distinct roles support a mechanism for phased commitment to invasion and can be targeted synergistically with invasion inhibitory antibodies.


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Parasite growth assays were analysed by by flow cytomentry on on an an Attune NxT flow cytometer. Live cell microscopy and immunofluorecence assays were performed using an an inverted microscope (Ti-E; Nikon) with NIS-Element Advanced Research imaging software (Nikon, V5.30) Microscopy images were processed and statistically analysed using the NIS Advanced Research software package (V5.30). Flow cytometry data were analysed using FACSDiva 6.1.3 software. Data resulting from live cell imaging experiments was statistically analysed using Prism (version 10).

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PKNH_1472300, PKNH_0623500, PKNH_0931500, and PKNH_1230100. All other data generated in this study are provided in the Supplementary Information/ Source Data file Sample sizes for each experiment were chosen to be consistent with the field norms. E.g., for flow cytometry experiments, at least three biological replicates, each with three technical replicates for each data point. FACS analyses to determine parasitaemia counts routinely measured the numbers of parasite-infected cells in a total of 50,000 red blood cells. Since parasitaemia values ranged from 0.1-15%, this was considered sufficient to provide statistically robust measurements of parasitaemia. For imaging, typically interactions resulting from 10-20 schizont egresses were analysed-e.g., as per 10.1371/journal.ppat.1004670. Specific values (e.g., number of merozoite-RBC contacts analysed per condition) are provided in the figures, figure legends and/or methods under the 'Statistics and Reproducibility' section.
No data were excluded from this study For all live cell imaging experiments, at least 2-3 independent repeats were performed per condition. For quantification of parasite replication by flow cytometry, 3-5 independent experiments, each with 3 technical repeats, were performed. For quantification of parasite growth by LDH assay, 2-3 independent experiments were performed. Westerns, PCRs, and IFAs demonstrating PkNBPXa and PvDBPa cKO status were repeated independently 3 times. For PkNBPXa and PkDBP secretion assays, 2 biological repeats were performed. For all experiments, all attempts at replication were successful.
Randomization was not relevant to this study as no subjective judgements were required about which data to include, exclude, or measure.
The investigators were not blinded during the experiment and/or when assessing the outcome. For growth assays, analysis were performed on quantitative endpoints that are subject to minimal investigator bias. Several live cell imaging experiments (eg. determining how many merozoites/schizont invaded BAPTA-treated red blood cells), also had clearly defined end points. For all other live cell imaging experiments, every effort was made to reduce investigator bias by identifying and clearly defining measurable, quantitative parameters (eg defining a deformation strength scoring system or quantifying the length of time a merozoite spent deforming a RBC) prior to the start of analysis and ensuring all relevant controls were in place. 2) Rat anti-HA is a commercially available antibody from: https://www.sigmaaldrich.com/GB/en/product/roche/roahaha? gclid=EAIaIQobChMIwaulgJPO-gIVA853Ch1MLAOkEAAYASAAEgIOMvD_BwE&gclsrc=aw.ds "Anti-HA High Affinity is a monoclonal antibody to the HA-peptide (clone 3F10). Anti-HA High Affinity recognizes the HA peptide sequence (YPYDVPDYA), derived from the influenza hemagglutinin protein. The antibody recognizes its antigenic determinant even when the HA peptide epitope is introduced into unrelated recombinant proteins by a technique known as "epitope tagging"." This antibody has been validated for detecting tagged proteins by western and IFA in P. knowlesi in the following publication: https:// doi.org/10.7554/eLife.45829

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3) Human anti-DARC is a commercially available antibody that binds specifically to the pentapeptide FEDVW of the Duffy receptor. Available from: https://absoluteantibody.com/product/anti-darc-2c3/Ab00893-10.0_human_igg1/bulk. This antibody has been validated by western blot and IFA. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of of group (a (a 'group' is is an an analysis of of identical markers).
All plots are contour plots with outliers or or pseudocolor plots.
A numerical value for number of of cells or or percentage (with statistics) is is provided. Tick this box to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information. Pk Pk A1-H.1 has been authenticated by by whole genome sequencing; Neither the HEK293E nor HEK293-6E cell lines were authenticated All lines tested negative for mycoplasma contamination

Methodology
No No commonly misidentified cell lines were used in in this study.
To To determine the multiplication rate of of mutant parasites, ring stage DMSO and rapamycin-treated cultures were adjusted to to a 0.5% parasitaemia and 2% 2% haematocrit and were grown in in triplicate in in 96 96 well plates in in a gassed chamber at at 37 37 degrees C. C. After 24 24 hours, a starting sample was taken and stained with SYBR Green I (1/5000 in in PBS; Life Technologies) before measuring parasitaemia by by flow cytometry (FACS). A second sample was taken 26 26 hours later, and a final sample was taken roughly 26 26 hours later again.
Data were collected on on an an Attune NxT flow cytometer using FACSDiva 6.1.3 software.
Data were collected using FACSDiva 6.1.3 software, and were analysed using FlowJo version 10. Graphpad Prism v10 was used for statistical analysis.
At At least 50,000 singlets were counted/sample. RBCs were gated by by plotting side scatter area against forward scatter area. Doublet discriminated was achieved by by gating forward scatter width against forward scatter height. Gating of of SYBR Green positive, infected RBCs was achieved by by plotting a histogram against BL1 (488 nm) height using a 530/30 filter. Parasitaemia was determined by by the number of of cells identified in in gate 3 as as a percentage of of those in in gate 2. 2.