Article Figures & Data
Figures
- FIG. 1.
Subcellular localization of HA9-tagged TgROM1 in intracellular parasites. (A) Model of the tagged construct of TgROM-1HA9. The protein is predicted to adopt a transmembrane topology with the N terminus (Nt) in the cytosol along with the epitope tag. The C terminus (Ct) is predicted to extend into the lumen. Catalytic triad residues (N, asparagine; H, histidine; and S, serine), are shown in transmembrane domains 2, 6, and 4). (B) IF localization of HA9-ROM1 driven by the endogenous promoter in transiently transfected parasites. HA9-ROM1 was partially colocalized with MIC2 at the tip of intracellular parasites but also extended throughout the apical half of the parasite. At 16 h posttransfection, cells were fixed, permeabilized, and incubated with anti-HA9 (α HA9) or anti-MIC2 (α MIC2) antibodies and revealed using secondary antibodies coupled to Alexa 594 (red), or Alexa 488 (green). IF images were processed by deconvolution microscopy, and a single Z-slice is shown in each example. Scale bar, 5 μm. (C) Transgenic parasites expressing HA9-TgROM1 under control of the TetOSAG4-regulatable promoter showed partial colocalization with MIC2 and MIC4, again extending throughout the apical end of the parasite. In contrast, MIC2 and MIC4 show almost perfect overlap at the apical border of the parasite. Cells were fixed; permeabilized; incubated with anti-HA9, anti-MIC2, or anti-MIC4 (α MIC4) antibodies; and revealed using secondary antibodies coupled to Alexa 594 (red) or Alexa 488 (green). IF images were processed by deconvolution microscopy, and a single Z-slice is shown in each example. Scale bar, 5 μm.
- FIG. 2.
Cryoimmuno-EM localization of TgROM1 in intracellular parasites. (A) Immuno-EM revealed that TgROM1 was localized in micronemes and the Golgi apparatus. Schematic representation of T. gondii showing dense granules (DG), rhoptries (ROP), micronemes (MIC), endoplasmic reticulum (ER), Golgi apparatus (Golgi), and nucleus (N). Cryoimmuno-EM was performed on extracellular parasites expressing HA9-ROM1 and detected with anti-HA9 antibody followed by secondary antibodies conjugated to 18-nm colloidal gold particles. Two separate images show the middle (a) and the apical end (b) of the parasite, respectively. Two other images taken at higher magnification show the Golgi apparatus (c) and the micronemes (d). Scale bars, 200 nm. (B) Quantitative distribution of immunogold from representative images of the Golgi apparatus and apical regions as defined in panel A. The distribution of TgROM1 was determined from 10 representative negatives stained as described above. The distribution of MIC2 was determined by staining with rabbit anti-C-domain followed by secondary antibodies conjugated to gold. Values shown are means ± standard deviations. (C) Double labeling with HA9-ROM1 (18-nm gold; large arrows) and MIC2 (12-nm gold; small arrows) revealed costaining of some compartments, while others contained only one of the markers. Scale bars, 200 nm.
- FIG. 3.
Localization of TgROM1 during cell invasion. (A) TgROM1 was found in apical compartments but was not detected at the surface of the parasite during invasion of host cell monolayers. Following a short invasion pulse of 5 min, monolayers were fixed and stained with antibodies to SAG1 (αSAG1) to reveal externally exposed epitopes. Monolayers were then permeabilized and incubated with anti-HA9 (αHA9) antibodies, followed by secondary antibodies coupled to Alexa 488 (green indicates SAG1) and Alexa 594 (red indicates HA9), respectively. IF images were processed by deconvolution microscopy. Arrowheads mark the site of apical attachment or the moving junction. Scale bars, 5 μm. (B) TgROM1 was not associated with the moving junction. Following pulse invasion, monolayers were fixed in 2.5% formaldehyde without detergent and incubated with anti-RON4 (αRON4) antibodies. Monolayers were then permeabilized and incubated with anti-HA9 antibodies followed by secondary antibodies coupled to Alexa 488 (green indicates RON4) and Alexa 594 (red indicates HA9). Scale bars, 5 μm. Arrowheads indicate the moving junction.
- FIG. 4.
Construction of TgROM1 knockdowns. (A) Schematic representation of the strategy used to obtain knockdowns in T. gondii. The construction of the Δrom1/S4HA9-ROM1 strain is shown as an example. The tTa strain was transfected with plasmid pS4HA9-ROM1 containing HA9-tagged TgROM1 expressed under the control of the TetOSAG4 promoter generating the ROM1/S4HA9-ROM1 strain (step 1). Recombinant ROM1/S4HA9-ROM1 parasites contained both the endogenous and the tagged copy of TgROM1. ROM1/S4HA9-ROM1 clones were then transfected with plasmid pΔR1YFP designed to delete TgROM1 by replacing the endogenous copy with the chloramphenicol resistance marker (cat) surrounded by 5′ and 3′ untranscribed regions and flanking regions of TgROM1 and driven by the SAG1 promoter (step 2). Parasites were subsequently cloned by limiting dilution to obtain the Δrom1/S4HA9-ROM1 strain. The chromosome of each strain is shown as a dashed line. (B) PCR analysis demonstrating the disruption of TgROM1. PCR was performed using genomic DNAs from ROM1/S4HA9-ROM1 and Δrom1/S4HA9-ROM1 strains as templates and primers described in panel A. Primer pairs F2-R2, F2-R3, and F4-R4 amplified fragments of the expected sizes only in the Δrom1/S4HA9-ROM1 strain. Primer pair F1-R1 amplified a fragment of the expected size only in the ROM1/S4HA9-ROM1 strain. The smaller band seen in the first lane in each case is a nonspecific product. PCR products were resolved on a 0.7% agarose gel stained with ethidium bromide. (C) TgROM1 transcripts were not detected in the Δrom1/S4HA9-ROM1 strain cultivated in the presence of Atc. ROM1/S4HA9-ROM1 and Δrom1/S4HA9-ROM1 strains were cultivated in the absence (−) or presence (+) of Atc. Total RNAs were extracted, and RT-PCR was performed using primers specific for TgROM. The single-copy ACT1 gene was amplified in parallel as a control (TgACT1). RT-PCR products were resolved on a 0.7% agarose gel stained with ethidium bromide. An arrow indicates the TgROM1 amplification product. The band seen at 0.9 kbp is a nonspecific amplification product. (D) Repression of HA9-TgROM1 by culture in Atc. Clones were incubated in the absence (−) or presence (+) of Atc for 16 h for IF and 48 h for Western blotting. Western blotting was performed on parasite lysates using anti-HA9 antibodies or MAb Tg17-43 against GRA1 as a loading control. The IF assay was performed using antibodies against MIC2 (α MIC2) and against the HA9 epitope (α HA9) to detect HA9-ROM1 in intracellular parasites. Scale bar, 2 μm.
- FIG. 5.
Competition assay demonstrating that TgROM1 was required for efficient growth of T. gondii. (A) Schematic representation of primers used for detection of the wild type (WT) and deleted ΔTgrom1 loci. Primers F1 and R1 selectively amplify the TgROM1 gene. Primers F5 and R5 selectively amplify the cat gene. (B) Δrom1 clones were outcompeted by strains containing an endogenous copy of TgROM1 in a mixed culture over time when the regulatable copy of HA9-ROM1 was repressed by continuous culture in Atc. In contrast, Δrom1 clones showed no growth defect when the regulatable copy was not repressed with Atc. Parasites were added at 1:1 at the starting time and cocultured in the absence (−) or presence (+) of Atc. Every 2 days, extracellular parasites were purified and genomic DNA was extracted for multiplex PCR analysis. ROM1/S1HA9-ROM1 and ROM1/S4HA9-ROM1 strains were tracked using primer pair F1-R1, and Δrom1/S1HA9-ROM1 and Δrom1/S4HA9-ROM1 strains were tracked using primer pair F5-R5. PCR products were resolved on a 1% agarose gels stained with ethidium bromide. (C) The intensity of the bands seen in panel B was quantified using a PhosphorImager. The ratios of the values obtained for the knockdowns in the Δrom1 background versus the nondeleted strains are shown for each passage in the absence (solid squares) or presence (solid diamonds) of Atc. The lines of best fit (dashed lines) were determined by linear regression.
- FIG. 6.
Decreased growth of TgROM1 knockdown clones was associated with slower growth and less-efficient invasion. (A) Monolayer lysis assay revealed a dose-dependent defect in the TgROM knockdown clones following suppression of ROM1. Monolayers of HFF cells were inoculated with Δrom1/S1HA9-ROM1 and Δrom1/S4HA9-ROM1 parasites in the presence (+) or absence (−) of Atc. After culture for 3 days, monolayers were rinsed, fixed, and stained with crystal violet and the absorbance was read at 570 nm. Decreased absorbance (staining of host cells) was a result of lysis due to parasite growth, as confirmed by microscopic examination. The x axis shows the challenge dose of parasites. Values are means ± standard deviations (n = 4 wells per sample), representative of four experiments. **, P < 0.001 by Student's t test. (B) Invasion assays revealed a modest reduction in invasion by TgROM1 knockdown clones following suppression of ROM1. Parasites were grown for several passages in the presence or absence of Atc, harvested from freshly egressed cultures, and used to challenge monolayers of HFF cells cultured on glass coverslips. Following a 5-min pulse invasion, parasites were classified as intracellular versus extracellular based on accessibility to staining of the surface antigen SAG1. Repression of ROM1 led to an ∼15% reduction in invasion in both the Δrom1/S1HA9-ROM1 and Δrom1/S4HA9-ROM1 strains. Values are means ± standard errors of the mean (n = 3 experiments). (C) Intracellular replication was more severely decreased in TgROM1 knockdown clones following suppression of TgROM1. Parasites were grown for several passages in the absence or presence of Atc, harvested from freshly egressed cultures, and used to challenge monolayers of HFF cells cultured on glass coverslips. Following 24 h of incubation, parasites were fixed and stained for the surface protein SAG and with DAPI to reveal nuclei, and the average number of parasites per vacuole was determined by counting under epifluorescence microscopy. Values are means ± standard deviations (n = 3 coverslips per group), representative of duplicate experiments. **, P < 0.001 by Student's t test.
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Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Summary of primers used in this study.
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- Supplemental file 1
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Summary of primers used in this study.
