Divergent roles for the RH5 complex components, CyRPA and RIPR in human-infective malaria parasites
Fig 4
Newly identified complex components PkRIPR, PkCSS and PkPTRAMP are essential for human erythrocyte invasion, as is PkCyRPA.
Inducible knockouts of pkcss, pkptramp and pkcyrpa were produced in the P. knowlesi DiCre expressing PkpSKIP 9–10 parasite line using CRISP/Cas9 gene editing. (A) PCR screen of wild type (pSKIP) and two clones of each transgenic parasite line (ptrampiKO, cssiKO, cyrpaiKO) showing successful and complete recombination of floxed gene sequences after rapamycin addition (+). Primer pair 4/5 specific for the ptramp locus amplified a 1741 bp band in wild type parasites, a 1993 bp band in mock -treated (-), transgenic parasites and a reduced size band of 965 bp after excision induced by rapamycin (+). Primer pair 10/11 specific for the css locus amplified a 1761 bp band in wild type, a 2013 bp band in mock-treated, transgenic parasites and a 919 bp band after excision. Primer pair 16/17 specific for the cyrpa locus amplified a 1467 bp band in wild type, a 1559 bp band in mock-treated and a 891 bp band in rapamycin-treated, transgenic cyrpaiKO parasites. DNA marker sizes are shown on the left. (B) Immunoblot of schizonts purified 26 h after DMSO (-) or rapamycin (+) treatment of ring stage cultures from wild type (pSKIP) and inducible knockout parasite lines. Two clones are shown for each knockout parasite. Blots were probed with anti-HA and anti-BiP antibodies as indicated on the right of the panels. All protein signals correspond approximately with their predicted molecular masses (HA-tagged PkCSS has a predicted molecular mass of 40.6 kDa, HA-tagged PkPTRAMP a predicted molecular mass of 39.2 kDa and PkCyRPA-HA a predicted mass of 42.5 kDa). Molecular mass standards are indicated on the left. (C) Invasion assay of wild type and inducible knockout parasites lines over one growth cycle. Parasitemias were measured by flow cytometry 40 h after rapamycin / DMSO treatment. Means and standard errors of three independent experiments carried out in triplicate are displayed. (D) Giemsa-stained brightfield images of wild type and inducible knockout parasites 30 h after DMSO (-) or rapamycin (+) treatment. Arrowheads point to merozoites attached to erythrocytes which have failed to invade. Scale bars equal 2 μm. (E) Growth inhibition assay of PkA1-H.1 parasites over one cycle with serial dilutions of purified IgG raised against recombinantly produced PkPTRAMP, PkCSS, PkRIPR or control rabbit IgG. Parasitemias were determined by flow cytometry 18 h after assay setup at schizont stage. Means and standard errors of three independent experiments in triplicate are displayed.