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. 2020 Oct 16;11:5258. doi: 10.1038/s41467-020-18991-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Schematic illustration of the constructs used for generation of the parasite expressing full-length GRA45 or GRA45 with indicated mutations. ACD and TL indicate predicted α-Crystallin domain and transthyretin-like fold, respectively. The conserved VxV or IxV motifs are indicated accordingly. b Extracellular Δgra45 parasites or Δgra45 parasites complemented with C-terminal HA-tagged wild-type or indicated mutant version of GRA45 were fixed with methanol and staining with indicated antibodies. The images are representative of results from two independent experiments (scale bar = 2 μm). c HFFs infected with indicated parasite strains for 24 h were fixed and stained with antibodies against HA epitope. The images are representative of results from two independent experiments (scale bar = 5 μm). d HFFs infected indicated parasite strains for 4 h were stained with antibodies against MAF1. The images are representative of results from two independent experiments (scale bar = 2 μm). e Δgra45 or Δgra45 complemented with wild-type or indicated mutant version of GRA45 were transiently transfected with GRA16-Ty (upper) or GRA24-Ty (lower) expressing plasmids and immediately used to infect HFFs and fixed at 24 h p.i. and subjected to the immunofluorescent assay with antibodies against Ty epitope. The images are representative of at least 40 host cells containing a single parasitophorous vacuole with four or more parasites (scale bar = 10 μm). f Murine BMDMs prestimulated with 5 ng/mL of IFNγ or left unstimulated for 24 h were infected with indicated parasite strains. Twenty-four hours p.i. parasite growth for each strain was measured by luciferase assay. Parasite growth in IFNγ-activated BMDMs is expressed relative to growth in naïve BMDMs. Data are displayed as mean ± SEM with independent experiments (n = 3) indicated by the same color dots. The significant difference between WT and knockout (or mutant strains) was analyzed with one-way ANOVA with Tukey’s multiple comparisons test and the p values are indicated above the columns.