Enhanced Nitrosative Stress during Trypanosoma cruzi Infection Causes Nitrotyrosine Modification of Host Proteins

Abstract

Oxidative/nitrosative stress may be important in the pathology of Chagas' disease. Experimental animals infected by Trypanosoma cruzi showed an early rise in myocardial and peripheral protein-3-nitrotyrosine (3NT) and protein-carbonyl formation that persisted during the chronic stage of disease. In comparison, experimental chronic ethanol-induced cardiomyopathy was slow to develop and presented with a moderate increase in oxidative stress and minimal to no nitrosative stress after long-term alcohol feeding of animals. The oxidative stress in both chagasic animals and animals with ethanol-induced cardiomyopathy correlated with the persistence of reactive oxygen species-producing inflammatory intermediates. Protein-3NT formation in T. cruzi-infected animals was associated with enhanced nitric oxide expression (inferred by nitrite/nitrate levels) and myeloperoxidase activity, suggesting that both peroxynitrite-and myeloperoxidase-mediated pathways contribute to increased protein nitration in Chagas' disease. We used one-and two-dimensional gel electrophoresis and Western blot analysis to identify disease-specific plasma proteins that were 3NT-modified in T. cruzi-infected animals. Nitrated protein spots (56 in total) were sequenced by matrix-assisted laser desorption ionization/time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry and identified by a homology search of public databases. Clustering of 3NT-modified proteins according to their functional characteristics revealed that the nitration of immunoglobulins, apolipoprotein isoforms, and other proteins might perturb their functions and be important in the pathology of Chagas' disease. We also showed that nitrated peptides derived from titin and ␣-actin were released into the plasma of patients with Chagas' disease. Such modified proteins may be useful biomarkers of Chagas' disease.

Increased Myeloperoxidase Activity and Protein Nitration Are Indicators of Inflammation in Patients with Chagas' Disease

“…For example, ONOO Ϫ -mediated increased protein 3-nitrotyrosine (3NT) formation is detected in the plasma and heart of mice infected by T. cruzi (13,27). We have found increased plasma and cardiac levels of protein carbonyls and malonyldialdehydes (MDA; lipid peroxidation markers) in mice and rats infected by T. cruzi (13,40).…”

“…For example, ONOO Ϫ -mediated increased protein 3-nitrotyrosine (3NT) formation is detected in the plasma and heart of mice infected by T. cruzi (13,27). We have found increased plasma and cardiac levels of protein carbonyls and malonyldialdehydes (MDA; lipid peroxidation markers) in mice and rats infected by T. cruzi (13,40). A substantial increase in the plasma level of MDA, in association with inefficient glutathione antioxidant defense, has been documented for seropositive patients with Chagas' disease (12,41).…”

A change of heart: oxidative stress in governing muscle function?

“…In addition, increased levels of phospholipid oxidation products, protein carbonylation, and a heightened glutathione antioxidant defence (Wen et al 2004) have been shown to associate with oxidative overload in Chagas' disease (Wen et al 2006b). Nitrated peptides derived from titin and actin are found in the plasma of patients with Chagas' disease, and may be useful as biomarkers for the disease (Dhiman et al 2008). In an experimental setting, oxidation of recombinant titin and actin enhanced their immunogenicity and recognition by sera antibodies from chagasic rats and patients (Dhiman et al 2012).…”

Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

“…The 2D gel analysis was performed as previously described (19). Briefly, the bacteria were lysed using lysis buffer (2 mol/L thiourea, 7 mol/L urea, 4% 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate, 2% dithiothreitol) supplemented with protein inhibitors.…”

“…S4 was incubated with 50 pg of rCST9 for 4 h; or MDM were exposed to S4 (MOI 40:1) and/or rCST9 for 2 or 20 h; then samples were processed for TEM as previously described (19). Briefly, hMDM or S4 were fixed in a mixture of 2.5% formaldehyde and 0.1% glutaraldehyde in 0.05 mmol/L cacodylate buffer (pH 7.2) containing 0.03% trinitrophenol and 0.03% CaCl 2 , washed in 0.1 mmol/L cacodylate buffer (pH 7.2), and postfixed in 1% OsO 4 in the same buffer.…”