Figure 1. RhopH2 and RhopH3 are membrane-associated proteins whose genes cannot be disrupted.
(A) Immunoblots showing that members of this complex are membrane-associated, but extractable by Na2CO3 (CO3); membr, integral membrane pool resistant to CO3. Only CLAG3 is cleaved by external pronase E, as indicated by an additional ~35 kDa band. (B) Immunofluorescent antibody (IFA) imaging of mature schizont-infected cells with indicated antibodies. Punctate labeling indicates that these proteins localize to rhoptry organelles in daughter merozoites. Scale bar, 5 µm. (C) IFA of trophozoite-infected cells, showing that each protein localizes to the host membrane and to the vacuole surrounding the intracellular parasite. Scale bar, 5 µm. (D) Matched-loading immunoblots from parasite stages shows production in schizonts (schz), incomplete delivery to invaded erythrocytes (ring- and trophozoite-infected cells), and release into culture supernatant (spent). CM, culture medium prior to cultivation is shown as a negative control. In each sample, proteins remain associated with membrane pellets (P) and are not soluble (S). (E) Schematic illustrates two plasmid transfection to produce knockouts (pL6-rhoph-KO), or transfection control that verifies sgRNA efficacy through repair with a codon-optimized version encoding an unmodified protein (pL6-rhoph-con). Streptococcus pyogenes Cas9 is expressed from pUF1-Cas9 (Ghorbal et al., 2014). (F) Ethidium-stained gel showing PCR-confirmed integration (Int) of positive control transfections (con) for each sgRNA on both genes, but retention of a wild-type site (WT) and pL6 episomes (P) in knockout transfections (KO).