Characterisation of a <i>gap45</i> KO.

<p>(<b>A</b>) Immunofluorescence analysis of <i>gap45</i> KO using indicated antibodies 72 hours after induction of loxP GAP45. Both MyoA and MLC1 are re-localised from the periphery of the parasite to the cytosol. Scale bars represent 5 μm. (<b>B</b>) Ultrastructural analysis of extracellular <i>gap45</i> KO parasites. (a) Low power of <i>gap45</i> KO extra-cellular tachyzoites showing the marked variation in shape from normal crescentic shaped parasites (arrow) to more spherical shape parasites (double arrows). The conoid of the parasites remains intact (C), rhoptries (R) and micronemes (M). Bar is 1 μm. (b) Detail of a spherical shaped organism showing the separation of the IMC from the plasmalemma disrupting the pellicular structure. R – Rhoptry. Bar is 1 μm. (<b>C</b>) Replication analysis of <i>gap45</i> KO parasites were performed 96 hours post-induction. Parasites were allowed to invade for 1 h prior replication for 24 h. To quantify the replication rate the number of parasites per parasitophorous vacuole was determined. Mean values of three independent assays are shown ± SEM (<b>D</b>) Trail deposition assays with indicated parasites were performed by allowing the parasites to glide on FBS coated cover slips for 30 minutes prior staining with α-Sag1 antibody. The assays were performed 96 hours post-induction ±50 nM rapamycin. Analysis of the <i>gap45</i> knockdown (Frenal et al., 2010) was performed after induction with ATc for 96 hours as described previously (Frenal et al., 2010). Scale bars: 20 μm. (<b>E</b>) Quantification of trail-deposition assays on indicated parasites shows no effect on the number of gliding trails in <i>gap45</i> KO parasites. Mean values of three independent experiments are shown, ± SEM. The number of trails was standardised to wild type parasites. (<b>F</b>) Kinetics of gliding motility of <i>gap45</i> KO parasites. The average speed of 19 manually tracked parasites was analysed over a time interval of 30 minutes. (<b>G</b>) Indicated parasites treated with and without 50 nM rapamycin 96 h prior performing egress assays. Intracellular parasites were artificially induced with Ca²⁺ ionophore (A23187) to trigger egress for 5 min. For quantification mean values of three independent egress assays are shown ± SEM. The egress rates were standardised to wild type parasites. (<b>H</b>) Invasion assays were performed 96 h after excision of <i>gap45</i>. Parasites were allowed to invade for 1 hour prior to washing the coverslips with 1× PBS to remove all extracellular parasites. Parasites were fixed after 24 hours and the amount of vacuoles in 25 fields of view were determined. Mean values of three independent assays are shown ± SEM. All strains were standardised to wild type parasites. (<b>I</b>) Immunofluorescence analysis of invading parasites demonstrates that parasites lacking <i>gap45</i> are capable of invading the host cell forming a tight junction (TJ). The TJ was visualised by staining against RON4. Scale bar: 5 μm.