Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates
Fig 5
Gene editing can delete PkP41 and PkGAMA in P. knowlesi.
Pkp41 and PkGAMA were replaced with eGFP to generate Pk41KO and PkGAMAKO strains respectively, as described in the Methods. A) Flow cytometry to establish eGFP expression in knockout lines. Enriched knock out and wild-type P. knowlesi cultures at late stages were labelled with SYBR green in 1X PBS and incubated for one hour after which they were quantified by flow cytometry. Events were gated as both SYBR green and GFP negative (lower left—uninfected RBCs), SYBR green only positive (lower right—RBCs infected with parasites not expressing GFP) and both SYBR green and GFP positive (upper right—RBCs infected with parasites not expressing GFP). Both KO lines expressed GFP, confirming integration of the KO cassette. B) eGFP expression in knock-out P. knowlesi strains as compared to wild-type P. knowlesi, imaged using fluorescence microscopy. Parasite nuclei were stained using Hoechst 33342; Merge is an overlay of eGFP and Hoeschst, confirming parasite expression of GFP. Scale bar is 2 micrometers. C) Proteins in knock out and wild-type P. knowlesi strains were localised using P. vivax polyclonal antibodies and Alexa Fluor 555 labeled secondary antibody then imaged using fluorescence microscopy. Localisation in both knock out and wild-type Pv41 and PvGAMA was performed using anti-Pv41 and anti-PvGAMA antibodies respectively; in both cases the KO line produced only diffuse background staining, confirming knockout of the P. knowlesi genes. Parasite nuclei were stained with Hoechst 33342; Merge is an overlay of Alexa 555 and Hoeschst. Scale bar is 2 micrometers.