ppat.1008049.g003.tif (1.22 MB)

Disruption of RAMA results in an invasion defect.

Download (1.22 MB)
posted on 06.09.2019 by Emma S. Sherling, Abigail J. Perrin, Ellen Knuepfer, Matthew R. G. Russell, Lucy M. Collinson, Louis H. Miller, Michael J. Blackman

(A) Reduced ring formation by RAMA mutants. Mature cycle 0 schizonts isolated from mock- or RAP-treated cultures of the indicated clones were incubated with fresh RBCs and ring-stage parasitaemia determined 4 h later by flow cytometry. Data were averaged from three biological replicate experiments, using blood from different donors. Error bars, ± 1 SD. Statistical significance was determined by a two-tailed unpaired t-test where p≤0.0001, **** and p>0.05, non-significant (ns). Ring formation by the parental 1G5DC parasites was unaffected by RAP treatment (t = 0.507, d.f. = 4, p = 0.639). (B) Still images from experiments monitoring egress of merozoites by time-lapse DIC microscopy of mature mock- and RAP-treated RAMAloxP clone 9C10 schizonts. Highly synchronous cycle 0 schizonts were incubated with the parasite cGMP-dependent protein kinase G inhibitor (4-[7-[(dimethylamino)methyl]-2-(4-fluorphenyl)imidazo[1,2-α]pyridine-3-yl]pyrimidin-2-amine (compound 2; C2) for 4 h to reversibly arrest egress [73]. Immediately after washing away the C2 the parasites were monitored for 30 minutes. No delay in egress was observed in the RAP-treated population, and there were no discernible differences in egress efficiency or morphology. Time in minutes from commencement of microscopy is indicated. Scale bar, 10 μm. Images shown are from clone 9C10 but are representative of 3 independent replicate experiments examining RAMAloxP clones 9C10 and 12C7. (C) Quantification of invasion, merozoite-induced RBC surface deformation and echinocytosis observed by video microscopy following rupture of mock- or RAP-treated RAMAloxP clone 9C10 schizonts. At least 20 videos per condition were quantified. Statistical significance was assessed by t-test; ns indicates not significant (p > 0.05) whereas **** indicates p < 0.0001. The RAP-treated (RAMAΔE2) merozoites interacted with and deformed target RBCs, but neither invaded them nor induced echinocytosis. See also S1 Movie and S2 Movie.