Linoleic acid is critical for sexual development in cultured cat cells

To determine the molecular mechanisms that define the species specificity of T. gondii sexual development, we generated cat intestinal organoids (Fig 1a) and then seeded these epithelial cells onto glass coverslips. These monolayers displayed intestinal epithelial properties, including polarization and tight junction formation (Fig 1b). To simulate natural infection, T. gondii was harvested from mouse brains 28–40 days after primary infection, and the parasites were released from the brain cysts by pepsin and acid digestion. After neutralization with sodium carbonate, parasites were seeded onto the cat intestinal monolayers, incubated for 5 days, and stained for markers of the parasite presexual stage called a merozoite [6,7]. Although we observed occasional dense granule protein 11B (GRA11B) and bradyzoite rhoptry protein-1 (BRP1) staining, the vast majority of the culture was negative for these merozoite markers (Fig 1c), suggesting that a required nutrient was limiting under these culture conditions. Because recent studies showed that the T. gondii asexual stages scavenge fatty acids, particularly oleic acid, from the host [8] and that sexual development of many fungi is dependent on linoleic acid [9], we surmised that supplementation with these fatty acids could facilitate T. gondii sexual development. We added 200 μM oleic or linoleic acid to cat intestinal monolayer culture medium 24 hours prior to infection with T. gondii. After 5 days of infection, we found that the addition of linoleic acid but not oleic acid caused approximately 35% of the T. gondii to express both merozoite stage markers (Figs 1d–1g and 2a, S1 Data). Similarly, GRA11B mRNA was significantly more abundant in cat intestinal cells supplemented with linoleic acid compared to any other condition (Fig 2b, S2 Data). As seen in vivo cat intestine, GRA11B changes localization from within the parasite dense granule organelles in the early stages of development to the parasitophorous vacuole and parasitophorous vacuole membrane in later stages of development [6]. We see similar localization of GRA11B depending on vacuole size, likely representing early, middle, and late stages (Fig 1e–1g). Likewise, BRP1 has a localization similar to that previously seen in the rhoptry organelles in the apical end of the merozoite [7] (Fig 1e–1g).

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larger image TIFF original image Download: Fig 1. Linoleic acid enhances progression through the sexual stages. (a) Cat intestinal organoids were generated from small-intestine sections and were grown in basement membrane matrix. Example of a growing organoid, 100-μm size bar. (b) Intestinal organoids were dissociated using trypsin and single cells seeded onto glass coverslips to grow as monolayers. The cells in the monolayer expressed the tight junction protein ZO-1 (green), 20-μm size bar. Cat intestinal monolayers were incubated with either (c) no fatty acid supplementation, (d) 200 μM oleic acid, or (e, f, g) 200 μM linoleic acid for 24 hours and then infected with ME49 bradyzoites for 5 days. Parasites undergoing presexual development were commonly seen only with linoleic acid supplementation as marked by staining with GRA11B (red) or BRP1 (green). Parasites in (e) early, (f) middle, or (g) late stages of sexual development were noted by differential localization of GRA11B. All panels are 20 μm2 with a 5-μm white size bar in the lower right corner. BRP1, bradyzoite rhoptry protein-1; DIC, differential interference contrast; GRA11B, dense granule protein 11B; ZO-1, zona occludens 1. https://doi.org/10.1371/journal.pbio.3000364.g001

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larger image TIFF original image Download: Fig 2. Quantification of merozoites in cat tissue culture. (a) Cat intestinal organoids were disassociated by trypsin and then grown as monolayers on glass slides. Slides were divided into three different groups: not supplemented with FA, supplemented with 200 μM OA, or supplemented with 200 μM LA. Monolayers were infected with T. gondii ME49 bradyzoites purified from brains of chronic infected mice at a 1:10 MOI. Five days after infection, staining for GRA11B and BRP1 along with DAPI allowed the percentage of vacuoles positive for GRA11B and BRP1 out of the total vacuoles to be determined. Total number of parasitophorous vacuoles were counted by positive DAPI staining and confirmed by morphology with DIC. Three biological replicates were counted, and on average, 35% of the total vacuoles were positive for both GRA11B and BRP1 in the LA-supplemented monolayers. *p-value = 0.0126 with N = 3 by two-tailed unpaired t test. Straining for both BRP1 and GRA11B was used to ensure that merozoite stages were counted. RNAseq and immunofluorescent imaging of the cat intestinal epithelium show that GRA11B is exclusively expressed in merozoites [6]. BRP1 is a rhoptry protein that was initially found in bradyzoites; however, it is also expressed in merozoites [7]. (b) Cat intestinal monolayers were grown as described in (a), except monolayers were quenched by TRIzol 5 days post infection, RNA was extracted, and cDNA was synthesized using an oligo (dT) primer to amplify mRNA. Expression of SAG1 and GRA11B were quantified by qPCR and the fold change calculated in comparison with uninfected cells. TUB1A was used to normalize gene expression across samples. GRA11B expression was significantly more abundant in the LA-supplemented monolayers with two biological replicates. *p-value = 0.0155 with N = 2 by two-tailed unpaired t test. BRP1, bradyzoite rhoptry protein-1; DIC, differential interference contrast; FA, fatty acid; GRA11B, dense granule protein 11B; LA, linoleic acid; MOI, multiplicity of infection; OA, oleic acid; qPCR, quantitative PCR; RNAseq, RNA sequencing; TUB1A, tubulin 1A; SAG1, surface antigen 1. https://doi.org/10.1371/journal.pbio.3000364.g002

Within the feline intestine, merozoites are known to differentiate into micro- and macrogametes that fuse to become diploid oocysts. After 7 days of infection, we saw round structures with reactivity to the macrogamete protein amine oxidase, copper-containing protein 2 (AO2) [10] in cat intestinal monolayers cultured with 200 μM linoleic acid but not in unsupplemented or oleic acid–supplemented cultures (Fig 3a–3c). PCR of these day 7 linoleic acid–supplemented cultures amplified message for AO2 as well as the predicted microgamete flagellar dynein motor protein TGME49_306338 with 44% identity to the homologue from the motile green alga Chlamydomonas reinhardtii (Fig 3d). In parallel, we assessed for the presence of intracellular oocyst wall biogenesis in these linoleic acid–supplemented cat cells by using the 3G4 antibody [11] that recognizes the T. gondii oocyst wall. There were approximately nine oocyst walls per cm2 of cultured cat cells supplemented with 200 μM linoleic acid but none in not-supplemented or oleic acid–supplemented cultures (Fig 3e–3h, S3 Data). Addition of 20 μM linoleic acid did not enhance oocyst wall production, indicating that the concentration of linoleic acid was critical for proper development.