Figure 5. MEDLE2 contains a host-targeting motif that is processed during export.

(A) Map showing the strategy used to engineer an ectopic copy of MEDLE2-HA in the thymidine kinase (TK) locus. Expression of an ectopic copy of MEDLE2-HA was driven by the MEDLE2 promoter. All point mutations were confirmed by Sanger sequencing (Figure 5—figure supplement 1). (B) Schematic representation of the MEDLE2 mutants generated using the strategy outlined in (A). The signal peptide (SP) is represented by dark blue, and low-complexity regions are shown in light blue. Candidate motifs targeted for mutagenesis are indicated with black triangles, and mutagenized amino acids are shown in red for two representative mutants. (C, D) Mutant parasites were used to infect HCT-8 cells and fixed for immunofluorescence assay (IFA) after 24 hr. For mutants shown in (C), the entire candidate motif was replaced with a matching number of alanine residues (e.g., KDVSLI/6A → AAAAAA). For mutants shown in (D), each individual amino acid in the KDVSLI sequence was changed to alanine. Red, hemagglutinin (HA)-tagged protein; green, parasites (VVL); blue, Hoechst. We note that SP and leucine 35 within the KDVSLI sequence are required for MEDLE2 export. (E, F) 5 × 10⁶ transgenic oocysts were used to infect HCT-8 cells for 48 hr before preparation of whole-cell lysates. Proteins were separated by for SDS-PAGE and analyzed by western blot. The resulting blots for infections with whole motif mutants (E) and individual amino acid point mutants (F) are shown. Red, neomycin; green, HA. Note that when mutants are expressed in mammalian cells and not C. parvum the resulting proteins do not show any size differences (Figure 5—figure supplement 2).

Figure 5—figure supplement 1. Sanger sequencing confirming the generation of MEDLE2 mutants.

100,000 sporozoites were used from each mutant strain for genomic DNA extraction. The resulting gDNA was used for PCR mapping of the thymidine kinase (TK) locus to verify the desired mutagenesis. 5′ TK–Nluc PCR products were used for TopoTA cloning, and the resulting colonies were grown and Sanger sequenced. Three colonies were sequenced for each strain using the M13 forward, M13 reverse, and an internal MEDLE2-specific primer to confirm targeted mutagenesis. The Benchling alignment of the Sanger sequencing result (transgene) to the reference sequence is shown. The black box highlights the mutation engineered in each strain.

Figure 5—figure supplement 2. MEDLE2 mutants are of the same size as wild type (WT) MEDLE2 when expressed in HEK293T cells.

Plasmids encoding human codon-optimized MEDLE2 with the N-terminal signal peptide removed (aa 2–20) (WT) or encoding the desired point mutations (denoted residues were replaced with alanines) were transfected into HEK233T cells. After 24 hr, whole-cell lysates were prepared, and proteins separated by SDS-PAGE for western blot analysis. MEDLE2 mutants were indistinguishable in size from WT MEDLE2. Red, neomycin; green, hemagglutinin (HA).

Figure 5—figure supplement 3. Uncropped images of panels shown in Figure 5D including infected and uninfected cells.

Transgenic C. parvum parasites with point mutations engineered in an ectopic copy of MEDLE2 expressed in the thymidine kinase (TK) locus were used to infect HCT-8 cells for 24 hr before fixation for immunofluorescence assay. L35A mutation results in accumulation of MEDLE2-HA (red) with the parasite (green).