Journal: Cell host & microbe

Article Title: The Chagas' disease parasite Trypanosoma cruzi exploits nerve growth factor receptor TrkA to infect mammalian hosts.

doi: 10.1016/j.chom.2007.05.006

Figure Lengend Snippet: Figure 2. TrkA-Dependent T. cruzi Invasion of Neuronal and Epithelial Cells (A) PC12WT cells, TrkA null mutant PC12nnr5cells, and PC12nnr5 cells stably expressing human TrkA (PC12TrkA) were infected with T. cruzi and, after 2 days, fixed and stained with DAPI and with human chagasic serum followed by anti-human IgG-Alexa 594 to visualize host cell nuclei and intracel- lular parasite, respectively, by fluorescence microscopy. (B and C) PC12WT, PC12nnr5, and PC12TrkA cells were infected with the indicated concentration of T. cruzi for 2 hr, and the degree of infection was estimated, after 2 days, by counting infected and noninfected cells (>300 cells) by phase-contrast microscopy after staining with Diff-Quik. (D) Infection ratio of mink lung epithelial cells (Mv1Lu) stably transfected with human TrkA gene (Mv1LuTrkA) over cells transfected with empty vector (Mv1LuEV) following a protocol analogous to the one for PC12 cells. Experiments were performed in triplicates and repeated at least three times with similar results. Error bars indicate the standard error of the mean.

Article Snippet: Mink lung epithelial cells (Mv1Lu) and the myoblast cell line L6E9 were purchased from ATCC.

Techniques: Mutagenesis, Stable Transfection, Expressing, Infection, Staining, Microscopy, Concentration Assay, Diff-Quik, Transfection, Plasmid Preparation

Journal: Cell host & microbe

Article Title: The Chagas' disease parasite Trypanosoma cruzi exploits nerve growth factor receptor TrkA to infect mammalian hosts.

doi: 10.1016/j.chom.2007.05.006

Figure Lengend Snippet: Figure 4. TrkA Kinase Inhibitors and Deletion of TrkA Intracellular Domain Block T. cruzi Invasion (A) K252a inhibits T. cruzi infection of PC12 cells. PC12WT cells were pretreated with 500 nM K252a for 1 hr prior to parasite addition. Infection assay is similar to that described in the Figure 2 legend. (B) K252a does not inhibit T. cruzi infection of PC12nnr5 cells; protocol is similar to that in (A). (C) AG879 inhibits T. cruzi infection of PC12WT but not of PC12nnr5 cells. Protocol is similar to that in (A) and (B) except for the pretreatment with 50 mM AG879. (C) IGF1-R inhibitor does not block T. cruzi infection of PC12WT cells. Protocol is similar to that in (A) except that cells were pretreated with several concentration of IGF-1R inhibitor and K252a. (E) Diagram of wild-type TrkA (TrkAWT) and TrkA lacking intracellular domain (TrkADICD). Tyrosine residues that become phosphorylated upon TrkA dimerization are missing in TrkADICD. (F) NGF (10 ng/ml, 48 hr) does not stimulate neurite extension in PC12nnr5 cells transfected with TkADICD (PC12-nnr5TrkA-DICD) or empty vector (PC12- nnr5EV), in contrast to the response of PC12nnr5 cells transfected with TrkAWT. After treatment, cells were fixed and stained with anti-neurofilament- 200 antibody and Alexa-conjugated secondary antibody. Scale bar, 10mm. (G) TrkAWT, but not TrkADICD, rescues T. cruzi invasiveness in TrkA null PC12nnr5 cells. Infection assays are similar to the protocol in (A)–(C), which show PC12-nnr5TrkA-WT cells supporting T. cruzi infection as well as PC12WT cells, in contrast to equally poorly permissive PC12-nnr5TrkA-DICD cells and PC12nnr5-EV cells. (H) TrkAWT, but not TrkADICD, enhances T. cruzi invasion of mink lung epithelial cells (Mv1Lu). Mv1LuTrkA-WT, Mv1LuTrkA-DICD, and Mv1LuEV cells were infected with the indicated concentrations of T. cruzi. Results were plotted as the ratio of Mv1LuTrkA-WT and Mv1LuTrkA-DICD over Mv1LuEV. The experiments in (A)–(D), (G), and (H) were repeated three times. The results are representative of one experiment of triplicate samples. Error bars indicate the standard error of the mean.

Article Snippet: Mink lung epithelial cells (Mv1Lu) and the myoblast cell line L6E9 were purchased from ATCC.

Techniques: Blocking Assay, Infection, Concentration Assay, Transfection, Plasmid Preparation, Staining