A phosphoproteomic approach towards the understanding of the role of TGF-ß in Trypanosoma cruzi biology.

Transforming growth factor beta (TGF-ß) plays a pivotal role in Chagas disease, not only in the development of chagasic cardiomyopathy, but also in many stages of the T. cruzi life cycle and survival in the host cell environment. The intracellular signaling pathways utilized by T. cruzi to regulate these mechanisms remain unknown. To identify parasite proteins involved in the TGF-ß response, we utilized a combined approach of two-dimensional gel electrophoresis (2DE) analysis and mass spectrometry (MS) protein identification. Signaling via TGF-ß is dependent on events of phosphorylation, which is one of the most relevant and ubiquitous post-translational modifications for the regulation of gene expression, and especially in trypanosomatids, since they lack several transcriptional control mechanisms. Here we show a kinetic view of T. cruzi epimastigotes (Y strain) incubated with TGF-ß for 1, 5, 30 and 60 minutes, which promoted a remodeling of the parasite phosphorylation network and protein expression pattern. The altered molecules are involved in a variety of cellular processes, such as proteolysis, metabolism, heat shock response, cytoskeleton arrangement, oxidative stress regulation, translation and signal transduction. A total of 75 protein spots were up- or down-regulated more than twofold after TGF-ß treatment, and from these, 42 were identified by mass spectrometry, including cruzipain-the major T. cruzi papain-like cysteine proteinase that plays an important role in invasion and participates in the escape mechanisms used by the parasite to evade the host immune system. In our study, we observed that TGF-ß addition favored epimastigote proliferation, corroborating 2DE data in which proteins previously described to be involved in this process were positively stimulated by TGF-ß.

Oral administration of GW788388, an inhibitor of transforming growth factor beta signaling, prevents heart fibrosis in Chagas disease.

BACKGROUND: Chagas disease induced by Trypanosoma cruzi (T. cruzi) infection is a major cause of mortality and morbidity affecting the cardiovascular system for which presently available therapies are largely inadequate. Transforming Growth Factor beta (TGFß) has been involved in several regulatory steps of T. cruzi invasion and in host tissue fibrosis. GW788388 is a new TGFß type I and type II receptor kinase inhibitor that can be orally administered. In the present work, we studied its effects in vivo during the acute phase of experimental Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: Male Swiss mice were infected intraperitoneally with 10(4) trypomastigotes of T. cruzi (Y strain) and evaluated clinically. We found that this compound given once 3 days post infection (dpi) significantly decreased parasitemia, increased survival, improved cardiac electrical conduction as measured by PR interval in electrocardiography, and restored connexin43 expression. We could further show that cardiac fibrosis development, evaluated by collagen type I and fibronectin expression, could be inhibited by this compound. Interestingly, we further demonstrated that administration of GW788388 at the end of the acute phase (20 dpi) still significantly increased survival and decreased cardiac fibrosis (evaluated by Masson's trichrome staining and collagen type I expression), in a stage when parasite growth is no more central to this event. CONCLUSION/SIGNIFICANCE: This work confirms that inhibition of TGFß signaling pathway can be considered as a potential alternative strategy for the treatment of the symptomatic cardiomyopathy found in the acute and chronic phases of Chagas disease.