Autocatalytic activation of a malarial egress protease is druggable and requires a protein cofactor (2021)
Figure 3

Figure 3. Efficient conditional disruption of MSA180.

(A) MSA180 architecture. Predicted signal peptide (black), conserved regions (hatched; see Appendix Fig S2 for sequence alignments), likely boundaries of the SERA6-interacting regions (double-headed arrows; fragments I and J from Fig 1B) and predicted molecular mass of each species based on its amino acid composition (in parentheses) are indicated. A semi-tryptic MSA180-derived peptide (filled block arrowhead) was detected from fragment I and is likely a result of SUB1 cleavage (see Fig EV4).

(B) Cas9-based approach for conditional disruption of the MSA180 gene. Sequential modification by marker-free gene editing was used to achieve: (I) insertion of a C-terminal HA3 epitope tag and a loxP site just downstream of the stop codon; followed by (II) insertion of a loxPint module just upstream of the central conserved region. Modification I produced a P. falciparum clone called MSA180-HA3 [B11], which was subsequently modified (II) to generate the MSA180-HA3:loxP parasite line. Conserved regions (black bars), sgRNA targeting sites (break), loxP sites (arrow heads), recodonised segments of MSA180 (hatched) and corresponding homology arms (regions linked by grey dotted lines) are shown. Half-arrows, primers used for diagnostic PCR to confirm gene editing and excision of floxed sequences (see Table EV1 for primer sequences).

(C) Diagnostic PCR confirming modification of the MSA180 locus in two independent MSA180-HA3:loxP P. falciparum clones.

(D) Diagnostic PCR confirming efficient DiCre-mediated disruption of MSA180 within the cycle of RAP-treatment (cycle 0) (representative of 6 independent experiments).

(E) Western blots of DMSO- and RAP-treated MSA180-HA3:loxP parasites showing successful epitope tagging and RAP-inducible ablation of MSA180-HA3 expression (red arrowhead; reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were subjected to SDS PAGE and blots probed with anti-HA and anti-MSA180 respectively. The anti-HA blot was reprobed with anti-AMA1 as a loading control. The numerous species detected by anti-MSA180 in RAP-treated MSA180-HA3:loxP extracts are likely non-MSA180-derived proteins cross-reactive with the polyclonal serum used.

(F) Representative IFA images confirming loss of MSA180-HA3 expression following RAP-treatment (reproducible in 2 independent experiments). Mature C2-arrested cycle 0 schizonts were co-stained with anti-HA (red) and anti-SERA5 (green). Merged signals include that of the DNA dye 4,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 5 μm.

(G) Western blots showing MSA180 is a predominantly soluble PV protein (representative of 2 independent experiments). Mature C2-arrested MSA180-HA3:loxP schizonts were sampled prior to (Total) and following saponin fractionation to separate RBC cytosol plus soluble PV proteins (Supernatant) from other parasite proteins and membrane-associated proteins (Pellet). The same samples were iprobed with anti-SERA5 and anti-MSP1 (mAb 89.1) as positive controls for soluble PV proteins and merozoite surface parasite proteins respectively. The presence of MSA180 p45-HA3 is likely a post-lysis artefact, perhaps due to SUB1 release during sample manipulation. Note the less efficient saponin-mediated release of MSA180-HA3 compared to SERA5, indicating limited membrane association of a fraction of MSA180. See also Fig EV3.