Trypanosoma cruzi Promotes Neuronal and Glial Cell Survival through the Neurotrophic Receptor TrkC▿

Trypanosoma cruzi, the agent of Chagas' disease, promotes neuron survival through receptor tyrosine kinase TrkA and glycosylphosphatidylinositol-anchored glial cell-derived family ligand receptors (GFRα). However, these receptors are expressed by only a subset of neurons and at low levels or not at all in glial cells. Thus, T. cruzi might exploit an additional neurotrophic receptor(s) to maximize host-parasite equilibrium in the nervous system. We show here that T. cruzi binds TrkC, a neurotrophic receptor expressed by glial cells and many types of neurons, and that the binding is specifically inhibited by neurotrophin-3, the natural TrkC ligand. Coimmunoprecipitation and competition assays show that the trans-sialidase/parasite-derived neurotrophic factor (PDNF), previously identified as a TrkA ligand, mediates the T. cruzi-TrkC interaction. PDNF promotes TrkC-dependent mitogen-activated protein kinase signaling, neurite outgrowth, and survival of genetically engineered PC12 neuronal cells and glial Schwann cells in a TrkC-dependent manner. Thus, TrkC is a new neurotrophic receptor that T. cruzi engages to promote the survival of neuronal and glial cells. The results raise the possibility that T. cruzi recognition of TrkC underlies regenerative events in nervous tissues of patients with Chagas' disease

A Novel Immunoprecipitation Strategy Identifies a Unique Functional Mimic of the Glial Cell Line-Derived Neurotrophic Factor Family Ligands in the Pathogen Trypanosoma cruzi▿

The journey of the Chagas' disease parasite Trypanosoma cruzi in the human body usually starts in the skin after an insect bite, when trypomastigotes get through the extracellular matrix to bind specific surface receptors in the epidermis and dermis to enter cells, where they differentiate and replicate. As the infection spreads to the heart, nervous system, and other parts of the body via the circulatory system, the parasite must also cope with additional receptors in the immune system and vascular endothelium. The molecular underpinnings that govern host cell receptor recognition by T. cruzi counterreceptors remain largely unknown. Here, we describe an immunoprecipitation strategy designed to concurrently identify host receptors and complementing parasite counterreceptors. Extracellular domains of growth factor receptors fused to human immunoglobulin G (IgG) Fc were incubated with parasite lysates, immunoprecipitated on protein G-Sepharose, and eluted with Laemmli sample buffer. Possible T. cruzi counterreceptors pulled down by the receptor-Fc bait were visualized on immunoblots probed with multispecific high-affinity IgG from chronic chagasic sera and on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels stained with silver or Coomassie blue. In screening receptors important for nervous system repair, this parasite counterreceptor immunoprecipitation (PcIP) assay identified 7 to 11 polypeptides (molecular masses, 14 kDa to 55 kDa) that bound to the coreceptors of glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) GFRα-1, -2, and -3. Binding was specific because the T. cruzi mimic of host GFLs, named TGFL, did not react with GFL coreceptor tyrosine kinase RET and with other neurotrophic receptors. The polypeptides were located on the parasite outer membrane and bound noncovalently to each other. TGFL eluted from the GFL receptor/protein G affinity column with 0.5 M NaCl, pH 7.5, and potently promoted neurite outgrowth and cell survival in a GFL-sensitive mouse pheochromocytoma cell line. Given that GFLs are neuron survival factors crucial for development and maintenance of central and peripheral nervous systems, it may be that T. cruzi mimicry of host GFLs helps in mutually beneficial host repair of infected and damaged nervous tissue. As there are >30 growth factor receptor-Fc chimeras commercially available, this PcIP assay can be readily adapted to identify receptors/counterreceptors in other T. cruzi invasion sites and in other infections such as Lyme disease, amebiasis, and schistosomiasis

Preferential Brain Homing following Intranasal Administration of Trypanosoma cruzi▿

The Chagas’ disease parasite Trypanosoma cruzi commonly infects humans through skin abrasions or mucosa from reduviid bug excreta. Yet most studies on animal models start with subcutaneous or intraperitoneal injections, a distant approximation of the skin abrasion route. We show here that atraumatic placement of T. cruzi in the mouse nasal cavity produced low parasitemia, high survival rates, and preferential brain invasion compared to the case with subcutaneously injected parasites. Brain invasion was particularly prominent in the basal ganglia, peaked at a time when parasitemia was no longer detectable, and elicited a relatively large number of inflammatory foci. Yet, based on motor behavioral parameters and staining with Fluoro-Jade C, a dye that specifically recognizes apoptotic and necrotic neurons, brain invasion did not cause neurodegenerative events, in contrast to the neurodegeneration in the enteric nervous system. The results indicate that placement of T. cruzi on the mucosa in the mouse nasal cavity establishes a systemic infection with a robust yet harmless infection of the brain, seemingly analogous to disease progression in humans. The model may facilitate studies designed to understand mechanisms underlying T. cruzi infection of the central nervous system

The Chagas' Disease Parasite Trypanosoma cruzi Exploits Nerve Growth Factor Receptor TrkA to Infect Mammalian Hosts

SummaryTrypanosoma cruzi, the agent of Chagas' disease, is an obligate intracellular parasite that invades various organs including several cell types in the nervous system that express the Trk receptor tyrosine kinase. Activation of Trk is a major cell-survival and repair mechanism, and parasites could use Trks to invade cells as a strategy to protect their habitat and prolong parasitism of vertebrate hosts. We show that T. cruzi binds to TrkA specifically and activates TrkA-dependent survival mechanisms. This interaction facilitates parasite adherence and promotes efficient invasion of neuronal, epithelial, and phagocytic cells via a process that requires TrkA kinase activity. Diffusible TrkA and TrkA-blocking agents neutralized infection in cellular and animal models of acute Chagas' disease, suggesting cellular receptors as therapeutic targets against parasitic diseases. Thus, TrkA, the nerve growth factor receptor commonly associated with neural survival and protection, may also underlie clinical progression of an important human parasitic disease

Intravenous administration of PDNF upregulates nerve growth factor in the heart selectively on cardiac fibroblasts.

<p><b>A</b>) Intravenous sPDNF upregulates NGF transcript in the heart of C57BL/6 mice. Mice (2–3/point) were injected with vehicle (PBS) or sPDNF intravenously (IV) (25 µg per mouse) daily for 2 or 6 days, sacrificed one day after the last injection, and their cardiac (atria) NGF transcript levels measured in duplicate by qPCR and compared to mice injected with IV PBS for 6 d. Results are representative of three similar experiments; *, P<0.05; ** P<0.01, ns, not statistically significant. <b>B</b>) Intravenous sPDNF upregulates NGF protein in the heart of C57BL/6 mice. Mice (2–3/point) were injected with sPDNF intravenously (25 µg per mouse) or vehicle for 6 d, sacrificed one day after the last injection, and their cardiac (atria) NGF protein levels ascertained by immunofluorescence using the NIH ImageJ software, n = 3–7, composite of two distinct experiments. <b>C</b>) NGF is preferentially localized in cardiac fibroblasts following intravenous injection of sPDNF. Representative images of heart (atria) tissue sections from the results presented in <b>B</b>) above. Note that iv sPDNF robustly increase NGF (leftmost panels) that co-localizes primarily with the cardiac fibroblasts (α-NGF/α-vimentin merge).</p

<em>Trypanosoma cruzi</em> Coaxes Cardiac Fibroblasts into Preventing Cardiomyocyte Death by Activating Nerve Growth Factor Receptor TrkA

<div><p>Rationale</p><p>Cardiomyocytes express neurotrophin receptor TrkA that promotes survival following nerve growth factor (NGF) ligation. Whether TrkA also resides in cardiac fibroblasts (CFs) and underlies cardioprotection is unknown.</p> <p>Objective</p><p>To test whether CFs express TrkA that conveys paracrine signals to neighbor cardiomyocytes using, as probe, the Chagas disease parasite <i>Trypanosoma cruzi</i>, which expresses a TrkA-binding neurotrophin mimetic, named PDNF. <i>T cruzi</i> targets the heart, causing chronic debilitating cardiomyopathy in ∼30% patients.</p> <p>Methods and Results</p><p>Basal levels of TrkA and TrkC in primary CFs are comparable to those in cardiomyocytes. However, in the myocardium, TrkA expression is significantly lower in fibroblasts than myocytes, and vice versa for TrkC. Yet <i>T cruzi</i> recognition of TrkA on fibroblasts, preferentially over cardiomyocytes, triggers a sharp and sustained increase in NGF, including in the heart of infected mice or of mice administered PDNF intravenously, as early as 3-h post-administration. Further, NGF-containing <i>T cruzi-</i> or PDNF-induced fibroblast-conditioned medium averts cardiomyocyte damage by H₂O₂, in agreement with the previously recognized cardioprotective role of NGF.</p> <p>Conclusions</p><p>TrkA residing in CFs induces an exuberant NGF production in response to <i>T cruzi</i> infection, enabling, in a paracrine fashion, myocytes to resist oxidative stress, a leading Chagas cardiomyopathy trigger. Thus, PDNF-TrkA interaction on CFs may be a mechanism orchestrated by <i>T cruzi</i> to protect its heart habitat, in concert with the long-term (decades) asymptomatic heart parasitism that characterizes Chagas disease. Moreover, as a potent booster of cardioprotective NGF <i>in vivo</i>, PDNF may offer a novel therapeutic opportunity against cardiomyopathies.</p> </div

Conditioned media from T cruzi-infected or sPDNF-stimulated cardiac fibroblasts protect cardiomyocytes against oxidative stress.

<p><b>A</b>) Conditioned media produced by T cruzi infection of cardiac fibroblasts confer protection of cardiomyocytes against H₂O₂-induced death in an NGF-dependent manner. Primary cultures of cardiomyocytes were exposed to 150 µM H₂O₂ for 4 h following preincubation with unconditioned medium from control fibroblast cultures (Un-CoM) or conditioned medium obtained by infecting cardiac fibroblasts with T cruzi (2×10⁵/mL, 24 h) (CoM). Cardiomyocytes were also placed in medium unexposed to cells, with or without 50 ng/mL NGF. Lastly, CoM was mixed with an α-NGF-blocking sheep antiserum (α-NGF) or normal sheep serum (NSS). Panel on the right shows representative staining from each condition. Data are combined from three independent experiments. <b>B</b>) Conditioned media produced by the specific stimulation of cardiac fibroblast by sPDNF confer protection of cardiomyocytes against H₂O₂-induced death in an NGF-dependent manner. Cardiomyocytes were exposed to 150 µM H₂O₂ for 4 h following preincubation with unconditioned medium (Un-CoM) or conditioned media obtained by stimulating cardiac fibroblasts with sPDNF (50 ng/ml, 3 h) (CoM). Cardiomyocytes were also exposed in parallel to CoM preincubated with an α-NGF-blocking sheep antiserum (α-NGF) or normal sheep serum (NSS). Data are combined from three independent experiments; * p<0.05, **p<0.01 ***p<0.001.</p

Cardiomyocytes preferentially expresses TrkA and cardiac fibroblasts TrkC.

<p><b>A</b>) TrkA levels are significantly higher in cardiomyocytes than in cardiac fibroblasts in tissue sections of the heart. <b>Left panel</b> shows the quantification of TrkA, vimentin (cardiac fibroblast marker), and myosin heavy chain (MHC, cardiomyocyte marker) identified by immunofluorescence on sections of C57BL/6 mouse hearts. Vimentin and MHC staining is plotted as function of TrkA fluorescence showing that TrkA is expressed by both cardiac fibroblasts and myocytes and that expression is higher in the myocytes. <b>Right panel</b> is a plot of vimentin and MHC mean fluorescence on pixels where TrkA is >200 (n = 3); ****, p<0.0001. <b>B</b>) TrkC levels are significantly higher in cardiac fibroblasts than on cardiomyocytes in tissue sections of the heart. <b>Left panel</b> shows the quantification of TrkC, vimentin, and MHC identified by immunofluorescence on sections of C57BL/6 mouse hearts, showing a preferential association of TrkC with cardiac fibroblasts. <b>Right panel</b> is a plot of vimentin and MHC mean fluorescence on pixels where TrkC is >200 (n = 3);****, p<0.0001.<b>C</b>) Visualization of the preferential expression of TrkA on cardiomyocytes in the heart. Representative images from the results presented in (<b>A</b>) above. Note preferential merge of TrkA and cardiomyocyte staining, scale bar = 100 µm. <b>D</b>) Visualization of the preferential expression of TrkC in cardiac fibroblasts in the heart. Representative images from the results presented in (<b>B</b>) above. Of note, TrkC staining merges preferentially in vimentin-stained cardiac fibroblasts, typically surrounding cardiomyocyte bundles, scale bar = 100 µm.</p

T cruzi infection upregulates NGF preferentially in primary cultures of cardiac fibroblasts compared to cardiomyocytes.

<p>A) T cruzi selectively upregulates NGF in cardiac fibroblasts. Primary cardiac fibroblasts (CFs) and cardiomyocytes (1°-CMs), and the cardiomyocyte cell line H9c2 were infected with T cruzi trypomastigotes (2×10⁵ per ml), and, at the indicated time points, their NGF mRNA was measured by qPCR (left panel). Similarly, culture supernatants were harvested and their NGF protein levels quantified by ELISA (right panel). Data are representative of three identical experiments; **, p<0.01, ***, p<0.001. B) Visualization of the preferential upregulation of NGF in cardiac fibroblast in T cruzi-infected co-culture of cardiac cells. Primary co-cultures (∼95% cardiac fibroblasts and 5% cardiomyocytes) were infected or not with 2×10⁵ T cruzi/mL for 24 h, then fixed. Leftmost panels: cells stained for NGF (red), and nuclei counterstained with DAPI (blue), scale bars = 100 µm. Right panels: high magnification of cells stained for NGF (red), vimentin (green), and MHC (blue), revealing upregulated NGF preferentially localized on cardiac fibroblasts in the T cruzi-infected co-cultures; scale bars = 10 µm.C) Cardiac fibroblasts secrete bioactive NGF in response to T cruzi infection. Primary cardiac fibroblasts were infected with T cruzi for 0 or 72 h and their conditioned media (CoM) were added to PC12 cell monolayers (left and center left panels). Note a robust neurite outgrowth produced by the 72-h CoM which was abolished by preincubating with a sheep antiserum against NGF (α-NGF) but not with normal sheep serum (NSS) (center right and right panels). Statistical significance is demonstrated in the bar-graphs, which represent the mean ± sd of >200 cells/well of triplicate wells, representative of three experiments; **, P<0.01; ***, P<0.001.</p

TrkA is targeted by T cruzi for upregulation of NGF in cardiac fibroblasts.

<p><b>A</b>) Antibodies against TrkA, but not against TrkB, TrkC or pan-neurotrophin receptor p75 (p75^NTR) significantly abrogate T cruzi-induced upregulation of NGF on cardiac fibroblasts. Primary cardiac fibroblasts were preincubated with the indicated antibodies (1 µg/ml), infected with T cruzi for 24 h, and the concentration of NGF in the culture overlays determined by ELISA. The results are the mean ± sd of triplicate points and represent the difference between NGF secreted by infected and uninfected cardiac fibroblasts; ***, P<0.001.<b>B</b>) shRNA against TrkA abrogates T cruzi-induced upregulation of NGF on cardiac fibroblasts. Cardiac fibroblasts were transfected with lentivirus encoding shRNA constructs against GFP, TrkA (two distinct vectors), or TrkC (one vector). Seven days later, fibroblasts were infected with T cruzi for 24 h, and NGF content in the culture overlays determined by ELISA. Results are the mean ± SEM of five separate experiments with similar results, and represent the difference between NGF secreted by infected and uninfected cardiac fibroblasts; *, P<0.05, **, P<0.005.</p