And 0.05 glutaraldehyde in PBS (two min), neutralized by 0.1 M glycine/PBS (5 min), and then blocked in 3 BSA/PBS (30 min). Noninvasive parasites or egressed vacuoles were stained with antiTgSag1 antibody (1:1,500, 1 hr) before detergent permeabilization. Cells have been washed 3x with PBS, permeabilized with 0.two triton X 100/PBS (20 min), and stained with antiTgGap45 antibody (1:3,000, 1 hr) to visualize intracellular parasites. L-Gulose site Samples have been washed and immunostained with Alexa488 and Alexa594conjugated antibodies (1:three,000, 1 hr). The amount of invaded parasites was deduced by immunostaining with antiTgGap45/Alexa594 (red), but not with antiTgSag1/Alexa488 (green). The egressed vacuoles were scored directly in the quantity of vacuoles with TgSag1stained parasites.Immunofluorescence LocalizationLocalization of epitopetagged proteins was performed by immunofluorescence assays. The system was basically exactly the same as described for invasion assays except for that samples have been permeabilized before incubation with antibodies. A panel of organellespecific antibodies (TgMic2 for micronemes, 1:1,000; TgRop1 for rhoptries, 1:1,000; TgGra5 for dense granules, 1:500; TgF1B for mitochondrion, 1:1,000; TgFd for apicoplast, 1:500; TgVP1 for acidocalcisomes/plantlike vacuole, 1:500) was employed collectively with antiHA antibody (1:five,000; SigmaAldrich, Germany) to assess localizations of epitopetagged PSS and PTS proteins. Images had been acquired applying ApoTome microscope (Zeiss, Germany).Functional Expression in E. coliThe M15/pREP4 strain was transformed with the empty pQE60 expression vector (Qiagen), pQE60TgPTS, pQE60TgPSS, or pQE60AtPSS [17] constructs and cultured in LuriaBroth medium supplied with ampicillin (one hundred mg/L) and kanamycin (50 mg/L). Protein expression was induced by 1 mM IPTG at 25 in overnight cultures containing five mM threonine or serine, followed by a 4 hr incubation at 37 . Lipids had been isolated and separated by onedimensional TLC in chloroform/methanol/acetate (130:50:20) and visualized by ninhydrin staining.PLOS Biology | DOI:ten.1371/journal.pbio.November 13,17 /Phosphatidylthreonine Is Required for the Parasite VirulenceLipid Extraction, TLC, and Phosphorus QuantificationParasites have been syringereleased from infected HFF (MOI, 3; 428 hrs of infection) and passed twice by way of 23G and 27G needles. Host debris was removed by filtering the parasite suspension by way of a 5 m filter (Merck Millipore, Germany). Cell pellets (0.51×108 parasites) have been resuspended in 0.4 ml of PBS and lipids have been extracted based on BlighDyer [41]. Briefly, 0.5 ml chloroform and 1 ml methanol have been mixed to the samples, which had been allowed to stand for 30 min and centrifuged (2,000 g, five min). The supernatant was transferred to a glass tube followed by addition of chloroform and 0.9 KCl (1 ml every single). Samples were mixed, centrifuged along with the reduce chloroform phase containing lipids was transferred to a conical glass tube. Samples had been stored at 20 in the airtight glass tubes flushed with nitrogen gas. Lipids have been resolved by twodimensional TLC on silica gel 60 plates (Merck) making use of chloroform/methanol/ ammonium hydroxide (65:35:five) and chloroform/acetic acid/methanol/water (75:25:5:two.two) because the solvents for the very first and second dimensions, respectively. They were visualized by staining with iodine vapors and identified according to their migration with authentic standards (Avanti Lipids). The main iodinestained phospholipid bands have been scraped off the silica plate, and quantif.
