Pius N. Nde

VNI Cures Acute and Chronic Experimental Chagas Disease

Chagas disease is a deadly infection caused by the protozoan parasite Trypanosoma cruzi. Afflicting approximately 8 million people in Latin America, Chagas disease is now becoming a serious global health problem proliferating beyond the traditional geographical borders, mainly because of human and vector migration. Because the disease is endemic in low-resource areas, industrial drug development has been lethargic. The chronic form remains incurable, there are no vaccines, and 2 existing drugs for the acute form are toxic and have low efficacy. Here we report the efficacy of a small molecule, VNI, including evidence of its effectiveness against chronic Chagas disease. VNI is a potent experimental inhibitor of T. cruzi sterol 14α-demethylase. Nontoxic and highly selective, VNI displays promising pharmacokinetics and administered orally to mice at 25 mg/kg for 30 days cures, with 100% cure rate and 100% survival, the acute and chronic T. cruzi infection.

Stable RNA interference of host thrombospondin-1 blocks Trypanosoma cruzi infection

Interactions between Trypanosoma cruzi and the extracellular matrix play an important role in cellular invasion. Here we show that T. cruzi increases the levels of thrombospondin‐1 (TSP‐1) expression in host cells during early infection. Stable RNA interference of host cell TSP‐1 knocks down the levels of TSP‐1 transcripts and protein expression in mammalian cells causing inhibition of T. cruzi infection. Addition of TSP‐1 to these cells restores infection. Thus, host TSP‐1, regulated by the parasite, plays a crucial role in early infection. This is the first report showing that a human parasite modulates TSP‐1 expression to facilitate infection.

Regulation and use of the extracellular matrix by Trypanosoma cruzi during early infection

Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global becoming a new worldwide challenge. For more than a century since its discovery, it has remained neglected with no effective drugs or vaccines. The mechanisms by which Trypanosoma cruzi regulates and uses the extracellular matrix (ECM) to invade cells and cause disease are just beginning to be understood. Here we critically review and discuss the regulation of the ECM interactome by T. cruzi, the use of the ECM by T. cruzi and analyze the molecular ECM/T. cruzi interphase during the early process of infection. It has been shown that invasive trypomastigote forms of T. cruzi use and modulate components of the ECM during the initial process of infection. Infective trypomastigotes up-regulate the expression of laminin γ-1 (LAMC1) and thrombospondin (THBS1) to facilitate the recruitment of trypomastigotes to enhance cellular infection. Silencing the expression of LAMC1 and THBS1 by stable RNAi dramatically reduces trypanosome infection. T. cruzi gp83, a ligand that mediates the attachment of trypanosomes to cells to initiate infection, up-regulates LAMC1 expression to enhance cellular infection. Infective trypomastigotes use Tc85 to interact with laminin, p45 mucin to interact with LAMC1 through galectin-3 (LGALS3), a human lectin, and calreticulin (TcCRT) to interact with TSB1 to enhance cellular infection. Silencing the expression of LGALS3 also reduces cellular infection. Despite the role of the ECM in T. cruzi infection, almost nothing is known about the ECM interactome networks operating in the process of T. cruzi infection and its ligands. Here, we present the first elucidation of the human ECM interactome network regulated by T. cruzi and its gp83 ligand that facilitates cellular infection. The elucidation of the human ECM interactome regulated by T. cruzi and the dissection of the molecular ECM/T. cruzi interphase using systems biology approaches are not only critically important for the understanding of the molecular pathogenesis of T. cruzi infection but also for developing novel approaches of intervention in Chagas disease.

Thrombospondin-1 Interacts with Trypanosoma cruzi Surface Calreticulin to Enhance Cellular Infection

Trypanosoma cruzi causes Chagas disease, which is a neglected tropical disease that produces severe pathology and mortality. The mechanisms by which the parasite invades cells are not well elucidated. We recently reported that T. cruzi up-regulates the expression of thrombospondin-1 (TSP-1) to enhance the process of cellular invasion. Here we characterize a novel TSP-1 interaction with T. cruzi that enhances cellular infection. We show that labeled TSP-1 interacts specifically with the surface of T. cruzi trypomastigotes. We used TSP-1 to pull down interacting parasite surface proteins that were identified by mass spectrometry. We also show that full length TSP-1 and the N-terminal domain of TSP-1 (NTSP) interact with T. cruzi surface calreticulin (TcCRT) and other surface proteins. Pre-exposure of recombinant NTSP or TSP-1 to T. cruzi significantly enhances cellular infection of wild type mouse embryo fibroblasts (MEF) compared to the C-terminal domain of TSP-1, E3T3C1. In addition, blocking TcCRT with antibodies significantly inhibits the enhancement of cellular infection mediated by the TcCRT-TSP-1 interaction. Taken together, our findings indicate that TSP-1 interacts with TcCRT on the surface of T. cruzi through the NTSP domain and that this interaction enhances cellular infection. Thus surface TcCRT is a virulent factor that enhances the pathogenesis of T. cruzi infection through TSP-1, which is up-regulated by the parasite.

Cellular Response to Trypanosoma cruzi Infection Induces Secretion of Defensin α-1, Which Damages the Flagellum, Neutralizes Trypanosome Motility, and Inhibits Infection

ABSTRACT Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that colonic epithelial model HCT116 cells respond to Trypanosoma cruzi infection by secreting defensin α-1, which reduces infection. We also report the early effects of defensin α-1 on invasive trypomastigotes that involve damage of the flagellar structure to inhibit parasite motility and reduce cellular infection. Short exposure of defensin α-1 to trypomastigotes shows that defensin α-1 binds to the flagellum, resulting in flagellar membrane and axoneme alterations, followed by breaking of the flagellar membrane connected to the trypanosome body, leading to detachment and release of the parasite flagellum. In addition, defensin α-1 induces a significant reduction in parasite motility in a peptide concentration-dependent manner, which is abrogated by anti-defensin α-1 IgG. Preincubation of trypomastigotes with a concentration of defensin α-1 that inhibits 50% trypanosome motility significantly reduced cellular infection by 80%. Thus, human defensin α-1 is an innate immune molecule that is secreted by HCT116 cells in response to T. cruzi infection, inhibits T. cruzi motility, and plays an important role in reducing cellular infection. This is the first report showing a novel cellular innate immune response to a human parasite by secretion of defensin α-1, which neutralizes the motility of a human parasite to reduce cellular infection. The mode of activity of human defensin α-1 against T. cruzi and its function may provide insights for the development of new antiparasitic strategies.

Early Regulation of Profibrotic Genes in Primary Human Cardiac Myocytes by Trypanosoma cruzi.

The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy. Examining the very early molecular events of T. cruzi cellular infection may provide disease biomarkers which will aid clinicians in patient assessment and identification of patient subpopulation at risk of developing Chagasic cardiomyopathy.

Epitope Capsid-Incorporation: New Effective Approach for Vaccine Development for Chagas Disease

Background: Previously we reported that a hexon-modified adenovirus (Ad) vector containing the invasive neutralizing epitope of Trypanosoma cruzi (T. cruzi) trypomastigote gp83 (Ad5-gp83) provided immunoprotection against T. cruzi infection. The purpose of this work was to design an improved vaccine for T. cruzi using a novel epitope capsid incorporation strategy. Thus, we evaluated the immunoprotection raised by co-immunization with Ad5-gp83 and an Ad vector containing an epitope (ASP-M) of the T. cruzi amastigote surface protein 2. Methods: Protein IX (pIX)-modified Ad vector (Ad5-pIX-ASP-M) was generated, characterized, and validated. C3H/He mice were immunized with Ad5-pIX-ASP-M and Ad5-gp83 and the cell-mediated responses were evaluated by enzyme-linked immunospot (ELISPOT) assay and intracellular staining. Immunized mice were challenged with T. cruzi to evaluate the vaccine efficacy. Results: Our findings indicate that Ad5-pIX-ASP-M was viable. Specific CD8+ T-cell mediated responses prior to the challenge show an increase in IFNγ and TNFα production. A single immunization with Ad5-pIX-ASP-M provided protection from T. cruzi infection, but co-immunizations with Ad5-pIX-ASP-M and Ad5-gp83 provided a higher immunoprotection and increased survival rate of mice. Conclusions: Overall, these results suggest that the combination of gp83 and ASP-M specific epitopes onto the capsid-incorporated adenoviruses would provide superior protection against Chagas disease as compared with Ad5-gp83 alone.

Phospho-proteomic analysis of primary human colon epithelial cells during the early Trypanosoma cruzi infection phase

The protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease, causes severe morbidity and mortality in afflicted individuals. About 30% of T. cruzi-infected individuals present with cardiac, gastrointestinal tract, and/or neurological disorders. Megacolon, one of the major pathologies of Chagas disease, is accompanied by gastrointestinal motility disorders. The molecular mechanism of T. cruzi-mediated megacolon in Chagas disease is currently unknown. To decipher the molecular mechanism of T. cruzi-induced alteration in the colon during the early infection phase, we exposed primary human colonic epithelial cells (HCoEpiC) to invasive T. cruzi trypomastigotes at multiple time points to determine changes in the phosphoprotein networks in the cells following infection using proteome profiler Human phospho-kinase arrays. We found significant changes in the phosphorylation pattern that can mediate cellular deregulations in colonic epithelial cells after infection. We detected a significant increase in the levels of phosphorylated heat shock protein (p-HSP) 27 and transcription factors that regulate various cellular functions, including c-Jun and CREB. Our study confirmed significant upregulation of phospho (p-) Akt S473, p-JNK, which may directly or indirectly modulate CREB and c-Jun phosphorylation, respectively. We also observed increased levels of phosphorylated CREB and c-Jun in the nucleus. Furthermore, we found that p-c-Jun and p-CREB co-localized in the nucleus at 180 minutes post infection, with a maximum Pearson correlation coefficient of 0.76±0.02. Increased p-c-Jun and p-CREB have been linked to inflammatory and profibrotic responses. T. cruzi infection of HCoEpiC induces an increased expression of thrombospondin-1 (TSP-1), which is fibrogenic at elevated levels. We also found that T. cruzi infection modulates the expression of NF-kB and JAK2-STAT1 signaling molecules which can increase pro-inflammatory flux. Bioinformatics analysis of the phosphoprotein networks derived using the phospho-protein data serves as a blueprint for T. cruzi-mediated cellular transformation of primary human colonic cells during the early phase of T. cruzi infection.