Journal: PLoS Biology

Article Title: Intestinal delta-6-desaturase activity determines host range for Toxoplasma sexual reproduction

doi: 10.1371/journal.pbio.3000364

Figure Lengend Snippet: Cat intestinal organoids were disassociated by trypsin and then grown as monolayers on glass slides. Monolayers were grown to confluency and then were incubated with either no fatty acid supplementation (a and e), 200 μM oleic acid (b and f), or 200 μM linoleic acid (c and g) for 24 hours and infected with ME49 bradyzoites purified from brains of chronic infected mice. After 7 days, monolayers were incubated with mouse anti-AO2 (a–c) or mouse monoclonal IgM 3G4 (e–g). The amiloride-sensitive AO2 is an enzyme exclusively expressed in macrogametes and early oocysts and has a possible role in oocyst wall biogenesis . AO2 expression was only detected by immunofluorescence in the monolayers supplemented with linoleic acid (c). 3G4 is a mouse monoclonal antibody produced by immunizing mice with purified oocyst walls ; thus, it is a marker of oocyst wall biogenesis. Only monolayers supplemented with linoleic acid (g) had positive 3G4 vacuoles. All panels are 20 μm 2 with a 5-μm white size bar in the lower right corner. (d) Markers for macrogamete and microgamete expression were also evaluated by PCR. Cat intestinal monolayers were grown in 24-well plates until confluency and then infected with T . gondii bradyzoites in duplicate using the same conditions as above. Seven days post infection, RNA was extracted with TRIzol, and cDNA was synthesized using an oligo (dT) primer to only amplify mRNA. AO2 was again used as a marker for macrogametes, and the expected PCR product is 218 bp. To assess microgamete presence, we selected the gene TgME49_306338, which is overexpressed in the gametes stage, corresponded to day 7 post infection in cats , and has 44% identity to a protein expressed in the flagella of the motile green algae C . reinhardtii . The expected PCR product for TgME49_306338 is 160 bp. TUB1A was used as an input control and results in a 172-bp product. “No RT” corresponds to a cDNA synthesis reaction without the addition of RT as a control for genomic DNA contamination. Equivalent amounts of cDNA per sample were used as a template for each PCR reaction, and the products were separated on an acrylamide gel. Bands with the correct size showing AO2 and TgME49_306338 expression were only observed in linoleic acid–supplemented monolayers. (h) The number of positive oocyst walls stained with 3G4 were quantified. Cat intestinal monolayers were infected with T . gondii bradyzoites and after 7 days, fixed with 3.7% formaldehyde in PBS, and incubated with 3G4 as showed in (e, f, and g). The number of positive oocyst walls were counted in each slide and divided by the area of slide in cm 2 . The number of positive oocysts walls in monolayers supplemented with linoleic acid was significantly higher than those supplemented with oleic acid in three biological replicates. * p -value = 0.0272 with N = 3 by two-tailed unpaired t test. AO2, amine oxidase, copper-containing protein 2; DIC, differential interference contrast; IgM, immunoglobulin M; RT, reverse transcriptase; TUB1A, tubulin 1A.

Article Snippet: Primary antibody was incubated at 4 °C overnight in 0.2% v/v Triton x-100 and 3% BSA in PBS (1:100 mouse anti-GRA11B, 1:100 rabbit anti-BRP1, 1:100 mouse anti-AO2, 1:50 monoclonal mouse anti-ZO-1 [Santa Cruz], or 1:25 mouse IgM anti-oocyst wall 3G4).

Techniques: Incubation, Infection, Purification, Expressing, Immunofluorescence, Produced, Marker, Synthesized, Algae, Control, cDNA Synthesis, Acrylamide Gel Assay, Staining, Two Tailed Test, Reverse Transcription