Plasmodium Protease ROM1 Is Important for Proper Formation of the Parasitophorous Vacuole
Figure 5
Pyrom1(-) salivary gland sporozoites have no defect in gliding, traversal, or host cell invasion.
(A) Salivary gland sporozoites from wildtype or the pyrom1 disrupted parasite line (R1INT) were allowed to glide on glass and gliding trails of deposited CSP were stained were visualized under fluorescent microscope (B) Quantification of sporozoite trails was carried out in triplicate and at least 25 fields were counted per well. The number of circular trails were quantified per sporozoite and shown is the mean percentage (± SD) of sporozoites that were associated with 0, 1–2, 2–10, or >10 circular trails. (C) Salivary gland sporozoites were loaded on top of a monolayer of Hepa 1–6 cells in the presence of 1 mg/ml FITC-dextran and allowed to traverse cells. As an internal negative control, sporozoites were treated with the microfilament inhibitor Cytochalasin D (CytD) prior to loading on top of Hepa 1–6 cells. The numbers of FITC-positive cells/field were counted under the microscope. The mean number of fluorescent cells/field (± SD) is shown for a representative. (D) Salivary gland sporozoites were loaded on Hepa1–6 monolayers and allowed to invade for two hours. A double staining assay was carried out to distinguish intracellular versus extracellular parasites. The % invasion was calculated as the number of (Green - Red parasites)/(Green parasites). Values presented are the mean number counts relative to the wildtype control (set to 1) ± standard deviation. (E) Kinetics of invasion was analyzed at three time points (10 min, 20 min, 40 min) during early invasion using the double staining assay described in (D). Y-axis values represent the % invasion of partially invaded or fully invaded parasites ± standard deviation.