Date Published: September 17, 2018
Publisher: Public Library of Science
Author(s): Shankar Suman, Girish Rachakonda, Sammed N. Mandape, Shruti S. Sakhare, Fernando Villalta, Siddharth Pratap, Maria F. Lima, Pius N. Nde, Helton da Costa Santiago. http://doi.org/10.1371/journal.pntd.0006792
Abstract: 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.
Partial Text: The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease which causes severe morbidity and mortality worldwide. Originally endemic in South American countries where it still constitutes a severe socioeconomic burden, Chagas disease has spread around the world and become a global health crisis [1, 2]. Currently, the disease is present in all major economically advanced countries due to modern globalization and migration . As many as 30% of afflicted individuals eventually present with cardiac, gastrointestinal tract and/or neurological disorders . The development of megacolon, as one of the pathologies of T. cruzi infection, is usually accompanied by unwanted changes in gastrointestinal (GI) tract motility which is thought to be due to decrease in the efficiency of the enteric nervous system [5, 6]. GI motility disorders have been attributed to alterations in the number of interstitial cells of Cajal and enteric nervous system defects. Although it is generally agreed that the enteric neurons [7, 8] and interstitial cells of Cajal [8, 9] decrease in numbers in megacolon, it is unclear what roles they play in the pathophysiology of chagasic megacolon. The presence of more natural killer and cytotoxic T-cells in colon lesions from patients with megacolon suggest that immune responses also play a role in the neuronal loss in chagasic megacolon patients . A study using a murine model of chagasic megacolon showed that megacolon was accompanied by increases in colon wall thickness, hypertrophy, and collagen deposition, which are hallmarks of fibrosis . This report correlates with others showing an increase in fibrotic lesions in smooth muscle and myenteric plexus of chagasic megacolon tissue sections . The fibrotic lesions observed in megacolon tissue sections can be caused by increased deposition of extracellular matrix (ECM) and matricellular proteins including TSP-1. The interactions between T. cruzi and colon cells including colon epithelium cells can deregulate cell signaling pathways leading to increased expression of transcription factors that upregulate the synthesis of ECM proteins [10, 11] causing fibrogenesis and cellular transformation reported in megacolon tissue sections. The role played by colon epithelium in the onset of chagasic megacolon remains unknown.
Chagasic megacolon is a major pathology associated with severe morbidity and mortality in T. cruzi-infected patients. The molecular mechanisms that cause megacolon in Chagasic patients remains largely undefined. Primary colonic epithelial cells constitute a good model for studying the mechanisms of T. cruzi infection of colon cells. We found that T. cruzi can infect colonic epithelial cells with more than 80% cellular infection after 72h (Fig 1). We hypothesize that during T. cruzi infection, the parasite deregulates host signal transduction and eventually the gene transcription profiles to cause symptoms associated with Chagas disease [10, 18, 19]. To elucidate the molecular mechanism of T. cruzi-induced molecular alterations in colon cells, we challenged primary human colonic epithelial cells with invasive T. cruzi trypomastigotes for various lengths of time (0, 60, 90, 120 and 180 minutes) to evaluate the pattern of altered phosphoproteins after infection using phospho-proteomic arrays and the mapping of biological network interactions. Analyzing the phosphorylation profiles of kinases and their protein substrates is essential for understanding how cells recognize and respond to the presence of T. cruzi in their micro-environment. Normal cellular activities in colon cells are highly susceptible to alterations following exposure to T. cruzi trypomastigotes. The analysis of phosphorylation profiles of kinases and their respective protein substrates may facilitate our understanding of the cellular response mediated by T. cruzi trypomastigotes in host colonic cells. In the present study, our goal was to decipher the phospho-signaling pathways that operate during early T. cruzi infection in human colonic epithelial cells. We investigated the phosphorylation activity of several axes of signaling cascades that may mediate T. cruzi-induced colon pathology. Our results showed that the parasite significantly altered the pattern of phosphorylated kinases and phosphoprotein levels during infection. Specifically, we found that T. cruzi deregulated the phosphorylated levels of 21 kinase phosphorylation sites, in the infected cells at different time points compared to uninfected controls. Bioinformatics analyses revealed that these phosphoproteins can be mapped to a variety of signaling pathways including those involved in neuronal, inflammatory, and fibrotic responses. Currently, it has been proposed that intense degenerative inflammatory changes occur in chagasic megacolon tissues, accompanied by increased collagen deposition and fibrosis. These changes are thought to mediate neuronal damage as well as intramuscular denervation, leading to chagasic megacolon . The bioinformatics network developed in our study revealed significant alterations in the phosphoproteome associated with the signaling pathways that may cause the colon pathology observed in chagasic patients. The altered phosphoproteins we observed in our study have been reported to play a role in cellular pathways that can cause tissue fibrosis, neuronal damages, and negative immunological responses. The upregulated phosphoproteins involved in neurological and inflammatory pathways can damage colonic myenteric neurons during T. cruzi infection . Bioinformatics analysis of array data also showed an overall increase in the proinflammatory response in HCoEpiC during T. cruzi infection. Hence, we further analyzed the associated proinflammatory pathways and found that NF-kB and JAK2-STAT1 pathways are activated by T. cruzi infection. The increased level of NF-kB (p-NF-kB, p-IKKα/β, p-TAK1 and p-IRAK4) revealed the high probability of activation of Toll like receptors by T. cruzi infection of HCoEpiC . The upregulated level of JAK2-STAT1 pathway in HCoEpiC also correlates with the activation of interferon signaling pathway during T cruzi infection. Our observation complements previous studies suggesting the activation of NF-kB and JAK-STAT signaling pathways by T. cruzi [22–24].