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. 2021 Dec 6;10:e70451. doi: 10.7554/eLife.70451

Figure 2. Infected cells express MEDLE2-HA across the parasite life cycle.

(A) 200,000 MEDLE2-HA-tdNeon transgenic parasites were used to infect HCT-8 cells and fixed at intervals across a 72 hr time period. Data shown are representative images from triplicate coverslips processed for immunofluorescence assay (IFA). Red, hemagglutinin (HA)-tagged protein; green, parasites (mNeon); blue, Hoechst. (B, C) Quantification of MEDLE2-expressing cells (red) versus intracellular parasites (blue) for 3695 host cells evaluated across a 72 hr time course. 20 fields of view quantified using ImageJ to identify host cells and parasites (B). The percentage of cell exhibiting MEDLE2-HA and mNeon staining is constant across the time course with a cumulative 94 ± 1.83% (mean ± SD) (C). (D) HCT-8 cultures infected with MEDLE2-HA parasites were fixed for IFA at 48 hr when sexual life stages were present. Cells were stained with stage-specific antibodies for female (COWP1) and male (α- tubulin), demonstrating MEDLE2 is exported across the parasite life cycle. Red, HA-tagged protein; green, parasites (stage-specific antibody); blue, Hoechst. (E) IFA of cryosections from the small intestine of Ifng-/- mice infected with MEDLE2-HA-tdNeon C. parvum (images representative of samples from three mice). Red, HA-tagged protein; green, parasites (tdNeon); blue, Hoechst; gray, Phalloidin (actin).

Figure 2—source data 1. Numerical data used for the quantification of HA positive host cells and the intracellular parasites.

Figure 2.

Figure 2—figure supplement 1. Construction of a MEDLE2-HA cytoplasmic tdNeon reporter parasite.

Figure 2—figure supplement 1.

(A) Schematic representation of the strategy to derive reporter parasite line in which MEDLE2 is 3× hemagglutinin (HA) epitope tagged and the parasite cytoplasm expresses a tandem mNeon green tag (tdNeon). The solid black arrow indicates the position of the Cas9-induced double-stranded break at the C terminus of the gene, which is the same guide used in Figure 1B to generate the MEDLE2-HA transgenic parasites. (B) PCR mapping modification of the MEDLE2 locus using genomic DNA from wild type (WT) and transgenic (MEDLE2-HA-tdNeon) sporozoites using the primer pairs shown in (A) and the thymidine kinase (TK) gene as a control. Note the presence of both a 1174 bp WT gene and a 4557 bp transgene. (C) HCT-8 cultures were infected with and MEDLE2-HA-tdNeon transgenic parasites and fixed at 24 hr. Red, HA-tagged protein; green, parasites (tdNeon); blue, Hoechst.