HMA in <i>Toxoplasma</i>-infected cells is strain-specific.

<p>(A–C) HFFs were labeled for 30 min at 37°C with 50 nM MitoTracker and then infected with (A) type I (RH), (B) type II (ME49), or (C) type III (CEP) strains of <i>T. gondii</i>. Cells were fixed 4 hpi and visualized by fluorescence microscopy. Phase, fluorescence (MitoTracker), and merged images are shown for each type. Scale bar, 5 µm. (D–F) Transmission electron micrographs depicting the PVM surrounding (D) type I, (E) type II, and (F) type III parasites grown in HFFs. Cells were fixed and processed for electron microscopy 4 hpi. Host mitochondria are indicated by M and parasites by P. (G) Percentage of the PVM associated with mitochondria in type I, II, and III vacuoles as determined by ImageJ analysis of electron micrographs (<i>n</i> = 20 for each). Values shown are mean ± SEM. ****<i>p</i><0.0001 using an unpaired <i>t</i> test.</p

Loss of HMA is associated with serum cytokine changes <i>in vivo</i>.

<p>(A) C57BL/6 mice (<i>n</i> = 5 per parasite strain) were infected subcutaneously with 100 type I or type I:<i>Δmaf1</i> tachyzoites and time to death assessed. (B) Mice (<i>n</i> = 4) were infected intraperitoneally with 100 tachyzoites of a type I or a type I:<i>Δmaf1</i> strain. At 5 dpi, PECs were isolated and supernatants analyzed for cytokine content at 6 and 12 h postisolation. (C) Sera (<i>n</i> = 12 per parasite strain) and (D) PECs (<i>n</i> = 3 per parasite strain) were isolated from mice infected intraperitoneally with 100 tachyzoites of a type I:<i>RFP</i>+ or type I:<i>Δmaf1:RFP+</i> strain. Values represent the ratio of the average MFI per cytokine in sera of type I/type I:<i>Δmaf1–</i>infected mice. Results from three independent experiments were pooled and values reported if <i>p</i><0.05 using an unpaired <i>t</i> test to compare average MFI values. PECs were permeabilized and labeled with APC/Cy7-conjugated CD11b antibody and processed for FACS analysis.</p

Exposure of the MAF1 C-terminus is essential for its role in HMA.

<p>(A) MEFs retrovirally transduced for expression of MAF1 were fixed, and following permeabilization, MAF1 was visualized by immunofluorescence microscopy using HA antibody, and host mitochondria were visualized using rabbit anti-TOM20 antibodies. Red arrowheads indicate MEFs expressing the transgene. Scale bar, 5 µm. (B) HFFs were co-infected with type I (white arrowhead) and type I parasites expressing C-terminally HA-tagged MAF1 (MAF1_CHA; green arrowhead) and fixed 4 hpi. Following permeabilization, MAF1 was visualized using anti-HA antibodies and mitochondria using anti-TOM20 antibody. Scale bar, 5 µm. (C) Percentage of PVM associated with mitochondria in HFFs 4 hpi with type I:MAF1 and type I:MAF1_CHA as determined by ImageJ analysis of electron micrographs (<i>n</i> = 23 for each). Values shown are mean ± SEM. ****<i>p</i><0.0001.</p

MAF1 induces changes in mitochondrial morphology and is associated with an altered host cell response to <i>Toxoplasma</i>.

<p>(A) Transmission electron micrograph of a mBMDM infected 17 hpi with type II:<i>MAF1</i> parasites. Cells were fixed and processed for electron microscopy. Scale bar, 0.5 µm. (B) Cross-sectional area of cytosolic and PVM-associated mitochondria during type I and type II:<i>MAF1</i> infection as determined by EM analysis (<i>n</i> = 20 for each). Values are mean ± SEM. ****<i>p</i><0.0001. (C) MEFs were mock-infected or infected with type II or type II:<i>MAF1 Toxoplasma s</i>trains at an MOI of 5. Then, 8 hpi, RNA was isolated from the cultures and analyzed using the Affymetrix Mouse 430 2.0 chip. Genes whose expression increased at least 2-fold during type II:<i>MAF1</i> infection relative to type II and were statistically significant at an FDR <5% for differential expression using SAM <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001845#pbio.1001845-Tusher1" target="_blank">[67]</a> are depicted. Colors indicate the deviation of each gene's signal above (yellow) and below (blue) its mean expression value across all six samples. (D) Primary mBMDMs were infected with type II, type II:<i>MAF1</i> strains, or mock-infected. Supernatants were collected at 17 hpi, passed through a 5 µm filter, and stored at −80°C. Samples were thawed on ice immediately prior to processing on the Luminex mouse 26plex (Millipore, USA) platform. Data shown are for the four cytokines with a MAF1-dependent difference and from three biological replicates (different days) ± s.e.m. Asterisks denote significant differences between mBMDM cells infected with type II versus type II:<i>MAF1</i>, *<i>p</i><0.05, ***<i>p</i><0.001, ****<i>p</i><0.0001 using a two-factor ANOVA analysis.</p

MAF1 (TGGT1_053770) mediates <i>Toxoplasma</i> HMA.

<p>(A) Type II or type II parasites expressing an N-terminally HA-tagged <i>TGGT1_053770</i> transgene were used to infect HFFs. Cells were fixed 12 hpi, and following permeabilization, TGGT1_053770 was visualized by immunofluorescence microscopy using polyclonal mouse sera raised against recombinant TGGT1_053770; host mitochondria were visualized using rabbit anti-TOM20 antibodies. (B) Transmission electron micrographs depicting the PVM surrounding type II and type II:<i>TGGT1_053770</i> parasites grown in mBMDMs. Cells were fixed and processed for electron microscopy 6 hpi. Host mitochondria are indicated by M. (C) Percentage of the PVM associated with mitochondria in HFFs 4 hpi with type I and type I:<i>Δmaf1</i>, and BMMs 17 hpi with type II and type II:<i>MAF1</i> vacuoles as determined by ImageJ analysis of electron micrographs (<i>n</i> = 20 for each). Values shown are mean ± SEM. ****<i>p</i><0.0001 using an unpaired <i>t</i> test. (D) Type I and type I:<i>Δmaf1</i> parasites (expressing RFP) were used to infect HFFs, which were then fixed 4 hpi. Following permeabilization, host mitochondria were visualized using rabbit anti-TOM20 antibodies. (E) Transmission electron micrographs depicting the PVM surrounding type I and type I:<i>Δmaf1</i> parasites grown in HFFs. Cells were fixed and processed for electron microscopy 4 hpi. Host mitochondria indicated by M. Scale bars are 5 µm (A and C) and 0.5 µm (B and D).</p

TGGT1_053770 is a novel secreted <i>Toxoplasma</i> protein and candidate mediator of HMA.

<p>(A) Schematic diagram of the TGGT1_053770 protein. Signal peptide (SP), TM domain, and phosphorylation sites (P sites) are indicated where predicted by SignalP 4.0 (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>), TMHMM v. 2.0 (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>), and ToxoDB (<a href="http://www.toxodb.org" target="_blank">www.toxodb.org</a>). (B) Microarray expression values (log2 ratios of sample intensity over control intensity) for 17 of the HMA-phenotyped progeny (black data points). Red, green, and blue data points represent expression values for type I (RH; HMA+), II (PDS; HMA⁻), and III (CTG; HMA+) strains, respectively. Data shown are averages across 10 probes on the cDNA expression array that map to the <i>MAF1</i> locus. (C) Lysates from extracellular type I parasites and type I–infected HFFs (intracellular) were treated with and without CIP and loaded in each lane. The membrane was probed with anti-HA antibody conjugated to peroxidase. (D) Normalized sequence coverage (<i>y</i>-axis) reflective of the number of copies of TGGT1_<i>053770</i> on chromosome II (<i>x</i>-axis) for types I (GT1), II (ME49), and III (VEG). The region putatively encoding TGGT1_053770 is indicated by a black bar. (E) Type I parasites expressing HA-tagged TGGT1_053770 were added to HFFs, and cultures were fixed 6 hpi. Following permeabilization, TGGT1_053770 was visualized using anti-HA antibodies. Scale bar, 5 µm. (F) Type I parasites were added to HFFs, and cultures were fixed 6 hpi. Following permeabilization, TGGT1_053770 was visualized using polyclonal anti-TGGT1_053770 mouse sera. Scale bar, 5 µm. (G) Western blot showing expression of TGGT1_053770 in type I, II, III and type II parasites. Blots were probed with antibodies to recombinant TGGT1_053770 (upper panels), then probed for surface antigen SAG1 as a loading control (lower panels); white vertical lines indicate that the order of the lanes shown is different from the original loading of the gel. Note that the product of the type III allele has a reproducibly slower electrophoretic mobility than type I. (H) Syringe-lysed parasites were plated on coverslips and fixed, labeled with anti-HA (TGGT1_053770) and anti-GRA7 antibodies, and visualized using confocal microscopy.</p

<em>Toxoplasma</em> Co-opts Host Cells It Does Not Invade

<div><p>Like many intracellular microbes, the protozoan parasite <em>Toxoplasma gondii</em> injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that <em>in vitro</em> the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting <em>Toxoplasma</em>-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where <em>Toxoplasma</em> encysts and persists. This phenomenon has important implications for how <em>Toxoplasma</em> globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.</p> </div

Uninfected-injected cells vastly outnumber Toxoplasma-mCherry-Cre cysts.

<p>Cre-reporter mice were infected with 5000 tachyzoites of Pru-mCherry-Cre parasites and sacrificed at 21 dpi. The brains were removed and sectioned in 40 <i>μ</i>m coronal sections which were then stained with DAPI and mounted on slides. The mounted sections were then imaged with a 10× objective on an epifluorescence microscope. Blue = DAPI, Green = ZsGreen, Red = mCherry. (a) A full coronal brain slice was reconstructed using a stitched grid of composite images. Scale bar = 1 mm (b) The boxed section of panel (a) was expanded to show the density of the ZsGreen⁺ cells. The arrowhead indicates a cyst within a host cell. Scale bar = 50 µm. (c) The boxed section of panel (b) was further enlarged to show the different morphologies and many fine processes of the ZsGreen⁺ cells. Scale bar = 50 µm.</p

Peritoneal exudate cells from Cre-reporter mice show U-I cells in mice infected with <i>Toxoplasma</i>-mCherry-Cre strain.

<p>Cre-reporter mice that express ZsGreen only after Cre-mediated recombination were inoculated i.p. with 5000 tachyzoites of either the control Pru-mcherry parasites (a) or the Pru-mcherry-Cre parasites (b,c). 4 days after inoculation, the peritoneal exudate cells (PECs) were removed, fixed, and examined by fluorescence microscopy. Panels from left to right show phase, red filter (parasite mCherry), green filter (host ZsGreen), and merge of the red and green filter images, respectively. Note the infected ZsGreen⁺ cell in (b) and the uninfected ZsGreen⁺ cell in (c). Scale bars = 10 µm.</p

Cultures of Cre-reporter cells infected with Toxoplasma-Cre strains show 2 distinct populations of uninfected-injected cells (U-I cells).

<p>Cre-reporter fibroblasts were incubated for 24 hours with <i>Toxoplasma</i>-Cre parasites at an MOI of 0.5, fixed and then examined by fluorescence microscopy. (a) Merged (red and green) image of a Pru-mCherry-Cre strain showing an infected (empty arrowhead) and adjacent, uninfected (arrow) eGFP⁺ host cell. Right panel is an enlargement of the boxed areas in the left panel. Red derives both from the DsRed of cells without Cre-mediated recombination and the mCherry expressed by the parasites. Green represents eGFP expression in a cell after Cre-mediated recombination. Scale bar = 50 µm for left panel and 10 µm for right panel(s). (b) As for (a) except uninfected eGFP⁺ cells that are not adjacent to infected cells are shown. (c) As for (a) except a host cell in the midst of division is shown. In this case, a non-mCherry expressing RH-Cre strain was used as the infecting strain and anti-SAG1 antibodies were used to visualize the parasites. The left panel shows a color merge where red represents both anti-SAG1 staining of the parasites and DsRed of the host cells and green derives from eGFP fluorescence; the right panel shows the corresponding phase image. Note the condensed nuclei (white filled arrowheads) of the dividing host cell. Scale bar = 10 µm.</p