Proteome Expression and Carbonylation Changes During Trypanosoma cruzi Infection and Chagas Disease in Rats

Abstract

Inflammation and oxidative stress, elicited by Trypanosoma cruzi infection, are important pathologic events during progressive Chagasic cardiomyopathy. In this study, we infected Sprague-Dawley rats with T. cruzi, and treated with phenyl-␣-tert-butylnitrone (PBN-antioxidant) and/or benznidazole (BZ-anti-parasite). We employed two-dimensional gel electrophoresis/mass spectrometry to investigate (a) the plasma proteomic changes associated with infection and disease development, and (b) the beneficial effects of PBN and BZ in controlling the disease-associated plasma profile. Matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) tandem MS (MS/MS) analysis of differentially expressed (total 146) and oxidized (total 48) protein spots yielded 92 unique proteins. Our data showed that treatment with PBN and BZ restored the differential expression of 65% and 30% of the disease-associated proteins to normal level, respectively, and PBN prevented development of oxidative adducts on plasma proteins. Western blotting to detect dinitrophenyl-derivatized carbonyl-proteins revealed plasma proteins were maximally oxidized during acute infection. Functional and disease/disorder analyses allocated a majority of the differentially expressed and oxidized proteins into inflammation/immunity and lipid metabolism categories and to molecular pathways associated with heart disease (e.g. cardiac infarction, contractile dysfunction, hypertrophy, and hypertension) in chagasic rats, and to curative pathways (e.g. ROS scavenging capacity, immune regulation) in infected rats treated with PBN and/or BZ. We validated the two-dimensional gel electrophoresis results by Western blotting, and demonstrated that the disease-associated increased expression of gelsolin and vimentin and release of cardiac MYL2 in the plasma of chagasic rats was returned to control level by PBN/BZ treatment. Increased plasma levels of gelsolin, MYL2 and vimentin were directly correlated with the severity of cardiac disease in human chagasic patients. Together, these results demonstrate the plasma oxidative and inflammatory response profile, and plasma detection of cardiac proteins parallels the pathologic events contributing to Chagas disease development,

Protein carbonylation as a major hallmark of oxidative damage: Update of analytical strategies

“…Significantly increased protein carbonylation levels were detected in the blood plasma of rats infected with Trypanosoma cruzi, of which 48 modified proteins were involved in transport (19%), inflammatory response (14%), complement activation (14%), metabolism (10%), tissue remodeling (10%), apoptosis (9%), acute-phase response (9%), DNA catabolic process (5%), myosin complex (5%), and response to cytokine stimulus (5%). Increased levels of oxidized proteins in specific biological processes, such as inflammatory response, complement activation, acute-phase, and cytokine response, in the course of infectious diseases supports the hypothesis of specific protein carbonylation (Wen & Garg, 2012). In a transgenic PS1 þ AbPP mouse model of Alzheimer's disease a total of 117 carbonylated proteins were identified by combining 2D Western blot analysis and MS. Pathway and network analysis revealed that three main networks were potentially altered during AD development: the iNOS-integrin signaling pathway, CRE/CBP transcription regulation and rab-lyst vesicular trafficking.…”

Innate Immune Responses and Antioxidant/Oxidant Imbalance Are Major Determinants of Human Chagas Disease

“…Two, increase in plasma LPO/MDA levels in seropositive/chagasic subjects may also be due to oxidatively-modified lipids released as a consequence of cellular injuries incurred in the cardiac tissue. This notion is supported by the observation of intense oxidative modifications of DNA, lipids and proteins in chagasic myocardium [13], [15] and identification of multiple oxidatively-modified cardiac proteins in sera/plasma of chronically-infected animals [27]–[28] and humans [18]. Three, SOD and GPX/GSH antioxidants, utilized by mammalian cells to cope with free radicals, were compromised in chagasic myocardium [14], [17].…”

Potential Role of Antioxidants as Adjunctive Therapy in Chagas Disease

“…Treatment with PBN controlled the pathologic oxidative tissue injury and preserved mitochondrial respiratory chain function in both acutely and chronically infected rodents. Wen and Garg [64] also showed that PBN restored the differential expression of 65% of the disease-associated proteins to the normal level and prevented the development of oxidative adducts on plasma proteins in chronically infected rats [51,64]. Importantly, PBN alone or in combination with benznidazole (but not benznidazole alone) preserved the left ventricular function that otherwise was significantly compromised in chronically infected Chagas rats [51,64].…”

“…PBN is a nitronebased antioxidant that scavenges free radical species and inhibits free radical generation [63]. The effects of PBN in CD have been reported in rats during the acute and chronic phases of infection using an oral administration of 1.3 mM of PBN for three weeks [51,64] and in mice during the acute phase with injection delivery of 50 mg/kg of PBN [50]. Treatment with PBN controlled the pathologic oxidative tissue injury and preserved mitochondrial respiratory chain function in both acutely and chronically infected rodents.…”