(A) Electron micrographs of intracellular WT (Δku80) (i and iii) and Δasp5 (ii and iv) tachyzoites within HFFs. Bars represent 1 µm (i,ii) and 200 nm (iii, iv). (i, ii) Low-power image showing WT (i) and Δasp5 (ii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), dense granules (D) and a Golgi body (G) located within a PV. Note the large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in wild-type parasites compared to Δasp5 parasites. (iii, iv) Details from the periphery of the PV showing a large host cell mitochondrion (HM) closely applied to the PVM in the wild type (iii) compared to the smaller mitochondrion (HM) associated with the Δasp5 PV (iv). (B) Quantitation of percentage of the PVM associated with host mitochondria, 5.59 ± 2.08% for Δasp5 parasites versus 24.3 ± 6.98% for wild-type parasites, mean ± standard error of the mean, P < 0.0001, n = 20 vacuoles. (C) (i) Mouse embryonic fibroblasts expressing MTS-GFP infected for 4 hr with wild type (Δhx), Δasp5CRISPR (a non-GFP positive knock out) or two independent ASP5 complemented clones (Δasp5CRISPR:ASP5WT-HA3). Localization of MAF1 at the PVM (top panel and bottom two panels) and mislocalized in intraparasitic puncta, potentially dense granules (panels 2 and 5), are shown in red. Mitochondria (MTS-GFP) are localized at the PVM in wild-type parasites (panel 1) and Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 (panels 5–6) to a large extent, but less so in the Δasp5CRISPR parasites (panels 2–4). (ii) Immunoblot using αHA antibodies against parasites expressing ASP5WT-HA3 and complemented mutants Δasp5CRISPR:ASP5WT-HA3 clones 1 and 2 shows the parasites express similar levels of HA-tagged ASP5 (as in Figure 2A), αGAP45 serves as a loading control. (D) Western blot of MAF1 species in wild-type and Δku80Δasp5 parasites. Blue arrow shows non-specific labeling (NS), αCatalase serves as a loading control. (E) Electron micrographs of intracellular wild type (i and ii) and Δku80Δasp5 (iii and iv) tachyzoites. Bars represent 1 µm (i, iii) and 200 nm (ii, iv). (i, ii) Low-power image showing wild-type (i) and Δasp5 (iii) tachyzoites containing a nucleus (N), rhoptries (R), micronemes (M), and dense granules (D) located within the PV. The large number of host cell mitochondria (arrowheads) associated with the PVM and the large NTN within the PV in the wild type compared to the Δasp5 parasites is noteworthy. (ii) Detail of the PV of a WT parasite showing the intertwining tubules of the NTN. HM – host cell mitochondrion. (iv) Detail of the PV surrounding a Δasp5 parasite showing granular material and a few vesicles (V) but absence of the tubular network. HM – host cell mitochondrion. Scale bar is 5 μm. ASP5, Aspartyl Protease 5; GFP, green fluorescent protein; HFFs, human foreskin fibroblasts; MTS, mitochondrial targeting sequence; NTN, nanotubular network; PV, parasitophorous vacuole; PVM, parasitophorous vacuole membrane; WT, wild type.