Characterization and Diagnostic Application of Trypanosoma cruzi Trypomastigote Excreted-Secreted Antigens Shed in Extracellular Vesicles Released from Infected Mammalian Cells.

Chagas disease, caused by Trypanosoma cruzi, although endemic in many parts of Central and South America, is emerging as a global health threat through the potential contamination of blood supplies. Consequently, in the absence of a gold standard assay for the diagnosis of Chagas disease, additional antigens or strategies are needed. A proteomic analysis of the trypomastigote excreted-secreted antigens (TESA) associated with exosomal vesicles shed by T. cruzi identified ∼80 parasite proteins, with the majority being trans-sialidases. Mass spectrometry analysis of immunoprecipitation products performed using Chagas immune sera showed a marked enrichment in a subset of TESA proteins. Of particular relevance for diagnostic applications were the retrotransposon hot spot (RHS) proteins, which are absent in Leishmania spp., parasites that often confound diagnosis of Chagas disease. Interestingly, serological screens using recombinant RHS showed a robust immunoreactivity with sera from patients with clinical stages of Chagas ranging from asymptomatic to advance cardiomyopathy and this immunoreactivity was comparable to that of crude TESA. More importantly, no cross-reactivity with RHS was detected with sera from patients with malaria, leishmaniasis, toxoplasmosis, or African sleeping sickness, making this protein an attractive reagent for diagnosis of Chagas disease.

Matrix metalloproteinase-2 degrades the cytoskeletal protein alpha-actinin in peroxynitrite mediated myocardial injury.

Matrix metalloproteinase (MMP)-2 mediates myocardial ischemia-reperfusion injury which is characterized by enhanced peroxynitrite biosynthesis during early reperfusion. Direct infusion of peroxynitrite into isolated hearts activates MMP-2 prior to the loss in mechanical function. The mechanical dysfunction is prevented by MMPs inhibitors. MMP-2 is also found in the sarcomere of cardiomyocytes where it cleaves troponin I and myosin light chain I. Cytoskeletal proteins such as alpha-actinin, desmin and spectrin are found in close association with the sarcomere and are known to be degraded in ischemia-reperfusion injury. It remains unknown whether these proteins are degraded in peroxynitrite-induced myocardial injury and if cytoskeletal proteins are also targets for MMP-2. Peroxynitrite (80 microM) was infused into isolated rat hearts which led to a delayed onset but rapidly developing decline in mechanical function. The MMPs inhibitor PD-166793 or the peroxynitrite scavenger glutathione prevented the decline in cardiac function. At the end of perfusion, alpha-actinin levels were decreased by 45+/-3% in peroxynitrite-infused hearts as compared to control hearts; however, this was normalized to that of control hearts with either PD-166793 or glutathione. Cardiac desmin and alphaII spectrin levels were unaltered following peroxynitrite infusion. alpha-Actinin and to a lesser extent desmin are susceptible to in vitro proteolysis by MMP-2 whereas spectrin is resistant. Cardiac dysfunction induced by peroxynitrite involves degradation of alpha-actinin that may be mediated by the proteolytic action of MMP-2.