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. 2022 Apr 11;13(2):e00623-22. doi: 10.1128/mbio.00623-22

FIG 3.

FIG 3

PfPMRT1 is essential for asexual blood stage development. (A) Simplified schematic of DiCre-based conditional PfPMRT1 knockout using selection-linked integration (SLI). Pink, human dihydrofolate dehydrogenase (hDHFR); gray, homology region (HR); green, T2A skip peptide; light blue, recodonized PfPMRT1; dark blue, 3×HA tag; yellow, neomycin phosphotransferase resistance cassette; orange, loxP sequence. Scissors indicate DiCre-mediated excision sites upon addition of rapalog. Stars indicate stop codons, and arrows depict primers (P1 to P5) used for the integration check PCR and excision PCR. (B) Diagnostic PCR of unmodified wild-type and transgenic condΔPMRT1 knock-in (KI) cell line to check for genomic integration using Primer P1-P4 as indicated in panel A. (C) Immunofluorescence assay (IFA) of condΔPMRT1 late stage schizont parasites showing localization of PfPMRT1-3×HA at the parasite plasma membrane (PPM) colocalizing with the merozoite surface protein 1 (MSP1). (D) Diagnostic PCR to verify the excision at the genomic level at 24 hpi/20 h post-rapalog addition for condΔPMRT1 and at 48 hpi for condΔPMRT1, c-nmd3PfPMRT1-ty1, and c-sf3a2PfPMRT1-ty1 parasites using primers P1 to P5 as indicated in panel A. Black arrowhead, original locus; red arrowhead, excised locus. (E) Western blot using anti-HA to verify knockout of PfPMRT1 on the protein level 4, 24, and 48 hpi. The expected molecular weight of PfPMRT1-3×HA is 53.3 kDa. Antibodies detecting aldolase and SBP1 were used as loading controls. (F) Growth curves of condΔPMRT1, c-nmd3PfPMRT1-ty1, and c-sf3a2PfPMRT1-ty1 parasites ± rapalog monitored over 5 days by flow cytometry. One representative growth curve is depicted (replicates in Fig. S5). The summary is shown as relative parasitemia values, which were obtained by dividing the parasitemia of rapalog-treated cultures by the parasitemia of the corresponding untreated ones. Shown are means ± SD from three (condΔPMRT1 and c-nmd3PfPMRT1-ty1) or four (c-sf3a2PfPMRT1-ty1) independent growth experiments. (G) IFA of condΔPMRT1 complemented with C-terminal TY1-tagged PfPMRT1 constructs expressed under either the constitutive nmd3 or the weak sf3a2 promoter to verify PPM localization. Scale bar, 2 μm.