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

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.

Thrombospondin-1 Plays an Essential Role in Yes-Associated Protein Nuclear Translocation during the Early Phase of Trypanosoma cruzi Infection in Heart Endothelial Cells

“…We showed that T. cruzi upregulates the levels of TSP-1 during the process of infection [19,34]. Others showed that TSP-1 is one of the downstream molecules of the hippo signaling cascade [22,25].…”

“…Furthermore, we also showed that the parasite interacts with host TSP-1 through T. cruzi calreticulin (TcCRT) located on the surface of the parasite [18]. The preincubation of host cells with an inhibitor of janus kinase (JNK) which is upstream of TSP-1, reduced the upregulation of TSP-1 induced by the parasite during the early phase of infection, indicating that TSP-1 upregulation is important and plays an essential role during infection [19]. The host cell signaling mechanism(s) regulated by the expressed host TSP-1 to facilitate infection and T. cruzi mediated pathogenesis remains to be elucidated.…”

“…We also showed that the expressed TSP-1 interacts with T. cruzi calreticulin (TcCRT) on the surface of the parasite, to facilitate cellular infection, which was inhibited in the presence of the TcCRT monovalent Fab antibody [18]. Furthermore, an increase in the cellular expression of TSP-1 induced by the parasite is essential for dysregulating the levels of phosphorylated proteins and cellular signaling [19].…”

Pyridinecarboxylic Acid Derivative Stimulates Pro-Angiogenic Mediators by PI3K/AKT/mTOR and Inhibits Reactive Nitrogen and Oxygen Species and NF-κB Activation Through a PPARγ-Dependent Pathway in T. cruzi-Infected Macrophages

“…It has been widely demonstrated that NF-κB is involved in the pro-inflammatory response in different models of T. cruzi infection (15,29,31,57,58). This transcriptional factor regulates the expression of several pro-inflammatory cytokines and chemokines such as TNFα, IL-1β, IL-6, and FIGURE 9 | Schematic representation of the pro-angiogenic and anti-inflammatory actions of HP 24 in T. cruzi-infected macrophages.…”

Differential Role of TGF-β in Extracellular Matrix Regulation During Trypanosoma cruzi-Host Cell Interaction

“…In T. cruzi -infected macrophage cultures, an increase in c-Jun phosphorylation was similarly observed [51]. Likewise, significant up-regulation of c-Jun was detected in early T. cruzi infection of primary human colonic epithelial cells by phosphoproteomics and Western blot [55]. In addition, primary cultures of cardiac myocytes displayed an increase in nuclear translocation of JunB up to 2 h after T. cruzi infection [56].…”