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. 2012 Apr 1;188(7):3404-15.
doi: 10.4049/jimmunol.1101425. Epub 2012 Mar 2.

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Sini Skariah  1 , Robert B Bednarczyk, Matthew K McIntyre, Gregory A Taylor, Dana G Mordue
Affiliations

Discovery of a novel Toxoplasma gondii conoid-associated protein important for parasite resistance to reactive nitrogen intermediates

Sini Skariah et al. J Immunol. .

Abstract

Toxoplasma gondii modifies its host cell to suppress its ability to become activated in response to IFN-γ and TNF-α and to develop intracellular antimicrobial effectors, including NO. Mechanisms used by T. gondii to modulate activation of its infected host cell likely underlie its ability to hijack monocytes and dendritic cells during infection to disseminate to the brain and CNS where it converts to bradyzoites contained in tissue cysts to establish persistent infection. To identify T. gondii genes important for resistance to the effects of host cell activation, we developed an in vitro murine macrophage infection and activation model to identify parasite insertional mutants that have a fitness defect in infected macrophages following activation but normal invasion and replication in naive macrophages. We identified 14 independent T. gondii insertional mutants out of >8000 screened that share a defect in their ability to survive macrophage activation due to macrophage production of reactive nitrogen intermediates (RNIs). These mutants have been designated counter-immune mutants. We successfully used one of these mutants to identify a T. gondii cytoplasmic and conoid-associated protein important for parasite resistance to macrophage RNIs. Deletion of the entire gene or just the region encoding the protein in wild-type parasites recapitulated the RNI-resistance defect in the counter-immune mutant, confirming the role of the protein in resistance to macrophage RNIs.

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Conflict of interest statement

Disclosures:

The authors have no financial conflicts of interest.

Figures

Figure 1
Figure 1
T. gondii CIM mutants display increased sensitivity compared to wild type parasites to the intracellular effects of macrophage activation. A. Parasite replication in naïve macrophages. B. Parasite replication or degradation following activation of infected macrophages with LPS and IFN-γ. C. Addition of aminoguanidine (1mM) to macrophages during activation to inhibit iNOS restores parasite replication. Quantification is the mean and standard deviation of two counts of 100 PVs each per conditions. Each experiments was performed a minimum of two times. Significance was determined by Student’s t-test. *Significance < 0.05; **Significance ** <0.01.
Figure 2
Figure 2
Representative pictures of WT parasites that have replicated to two or four parasites per vacuole compared to the 2B6 mutant that either remained at one parasite per vacuole (static) or appeared amorphous/degraded (bottom panel) Parasites were stained with a polyclonal antibody against Toxplasma gondii.
Figure 3
Figure 3
The impaired replication/survival of T. gondii CIM mutants is not dependent on macrophage Irgm1/Irgm3. A. Parasite replication in naïve macrophages; B. activated macrophages; C. activated Irgm1/3 macrophages. Quantification is the mean and standard deviation of two counts of 100 PVs each per conditions. The experiment was performed once. Significance was determined by Student’s t-test. *Significance < 0.05; **Significance ** <0.01.
Figure 4
Figure 4
Nitric oxide donors in the absence of macrophage activation are sufficient to impair replication/survival of many of the CIM mutants. A. Infected macrophages were cultured in the presence of 100μM/ml sodium nitroprusside (SNP) for 24 hours. B. Infected macrophages were cultured in the presence of 100μM/ml DETA NONOate for 24 hours. Quantification is the mean and standard deviation of two counts of 100 PVs each per conditions. Each experiments was performed a minimum of two times. Significance was determined by Student’s t-test. *Significance < 0.05; **Significance ** <0.01.
Figure 5
Figure 5
The defect in the CIM mutants is not associated with increased macrophage iNOS levels. A, B. Qualitative and quantitative analysis of iNOS levels in infected macrophages by fluorescent microscopy. Arrowheads point to infected macrophages that have increased iNOS expression. Parasites without arrowheads are in macrophages that have not increased iNOS expression following activation. C. Quantification of iNOS transcript by qRT-PCR. Fold change increase in iNOS transcript levels in macrophages with and without parasites following activation relative to naïve macrophages. The experiment was repeated twice. Normalization was performed with GAPDH. Lines indicate a significance value of <0.05 between naïve and activated macrophages but not between any of the activated samples. No bands were present in the absence of reverse transcriptase. D. Quantification of iNOS protein in macrophages with and without parasites relative to mouse actin. The experiment was repeated twice. 2B6 Recap and 2B6 deletion are deletions that recapitulate the original plasmid insertion (2B6 Recap) or have a targeted deletion of the 2B6 protein (2B6 deletion).
Figure 6
Figure 6
TGME49_010810 gene encodes a transcript and results in an expressed protein A. Schematic of the transcript for the two isoforms of TGME49_010810 gene Untranslated (UTR) regions are darkly shaded, exons are represented by white boxes and introns as solid lines. The plasmid insertion site relative to the transcript is shown as an arrow. The region deleted in the gene deletion clone is shown. B. cDNA expression of the 2B6 transcript relative to actin in WT versus gene deleted parasites. The 2B6 mutant, Recap 1 (R1), Recap 2 (R2) and the 2B6 deletion of just the protein region (deletion) no longer express 2B6 cDNA. No bands were present in the absence of reverse transcriptase (data not shown). C. Western blot of wild type and 2B6 mutant parasites using a polyclonal antibody created to recombinant TGME49_010810 protein. T. gondii surface associated antigen-1 (SAG1) expression is shown as a loading control and was detected with the mAb DG52. The experiment was done twice. D. Western blot analysis of the 2B6 protein relative to SAG1 in WT parasites versus the 2B6 mutant, 2B6 Recap clone and 2B6 targeted protein deletion.
Figure 7
Figure 7
Replication of T. gondii ΔTGME49_010810 clones compared to WT parasites in A. naïve macrophages or B. following macrophage activation. Parasites with the entire TGME49_010810 cDNA disrupted (Recap 1 and 2) or parasites with just the protein coding region deleted (Deletion 1) recapitulate the phenotype of the 2B6 mutant. The mean and standard deviation of two counts of 100 PVs each is shown. Each experiment was performed at least twice.
Figure 8
Figure 8
Immunolocalization of an endogenous YFP tag of the TGME49_010810 protein in intracellular and extracellular parasites. A. Expression of the YFP-tagged 2B6 protein in WT versus the 2B6-YFP parasite clone (left). Western blot analysis of YFP protein of WT parasites compared to the 2B6-YFP parasite clone (right). B. Localization of the endogenous TGME49_010810-YFP protein relative to the apical ISP-1 protein and the parasite nucleus (DAPI). The merge image is also stained with 594 ani-rabbit Toxoplasma antibody to mark the parasite (light red). C. Localization of TGME49_010810-YFP to the conoid following induction of conoid extrusion with ethanol. Lower panel shows a magnification of a single parasite with the conoid extruded and TGME49_010810-YFP expression. D. Quantification of the number of parasites that expressed the TGME49_010810-YFP in the conoid plus perenuclear, the conoid only, the perinuclear only, the conoid plus perinuclear plus basel end of the parasite or in the basel end of the parasite alone. TGME49_010810-YFP was expressed in the cytoplasm of the parasite in all of the parasites (B and C and data not shown). Each experiment was performed at least twice.
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