Fig. 4: Immunodominant antigen 23 is essential and required for reinvasion of host cells.

a, b HCT8 cell monolayers infected with Cp23-diCRE in the presence or absence of rapamycin. At 6, 24, and 48 h, gDNA was extracted and diagnostic PCRs confirmed the level of excision at the given timepoint. Data shown is representative of 2 biological replicates. c HCT8 cell monolayers infected with Cp23-diCRE in the presence or absence of rapamycin. At 6, 24, and 48 h, the monolayer was fixed and stained and the parasite per host nuclei was quantified. Box plot whiskers show the minimum and maximum values, the box shows the 25th to 75th percentile and the line shows the median. Representative images are shown, with nuclei in blue (Hoechst), and parasites in green (Vicia villosa lectin). Scale bar = 30 μm. Data shown is representative of two biological replicates. Significance was determined using a two-tailed unpaired t test. d. Ifnγ^–/– mice were infected with 50,000 Cp23-diCRE parasites and treated with rapamycin or DMSO control in their drinking water at 2 days post infection. Data shows the mean faecal luminescence from a pooled cage sample ( ± SEM of 2 technical replicates), n = 2 mice per condition. e Super resolution microscopy at 24 h post infection in vitro with Cp23-diCRE parasites in the presence or absence of rapamycin; green (helix pomatia agglutinin (HPA)), parasite; magenta, (sytox), nuclei; red (Cp23 Ab), Cp23. Scale bar = 2 μm. Data shown is representative of two biological replicates. f Expansion microscopy of the C. parvum asexual stages: sporozoite, merozoite, trophozoite and meront, and the sexual stages: macrogamont (female) and microgamete (male); green (helix pomatia agglutinin (HPA)); blue (N-hydroxysuccinimide (NHS ester); magenta (sytox); red (αCp23). Scale bar = 5 μm. Median expansion factor of 4.5. g Transmission electron microscopy of an excysted and unexcysted C. parvum sporozoite coupled with immunogold labelling with Cp23 antibody. Scale bar = 300 nm. h Permeabilisation assay of C. parvum sporozoites. The permeabilised condition used 0.1% Triton X-100 and the non-permeabilised condition used PBS. Grey (tryptophan synthase (αTrpB); green (Vicia villosa lectin); red (αCp23); blue (Hoechst). All images were taken using the same settings and exposure. Scale bar = 5 μm. Data shown is representative of two biological replicates. i HCT8 cell monolayers infected with either Cp23-diCRE or wildtype parasites in the presence or absence of rapamycin, and at 22 h post infection life stages were quantified. Data shows 2 biological replicates ± sd. n = 438 Cp23-diCRE - RAP, n = 173 Cp23-diCRE + RAP, n = 213 wildtype—RAP, n = 203 wildtype + RAP. j, k Live imaging of the reinvasion event was carried out at 18 h post infection of an HCT8 monolayer with Cp23-diCRE parasites in the presence or absence of rapamycin. All live microscopy data shown is from 2 biological replicates with n = 9 egress events recorded for each condition. Gliding distance was only measured for merozoites with clear movement following egress (n = 18 for DMSO and n = 5 for RAP). Movement of individual merozoites was tracked using Fiji software. Scatter plots show the mean ± sd with significance determined using a two-tailed unpaired t-test. Representative images are shown in (k) with movies in supplementary data. Scale bar = 8 μm. Note that a cell culture dish in the figure indicates an in vitro experiment, while a mouse silhouette indicates an in vivo experiment.