Abstract
Trypanosoma cruzi (Tc) infection causes Chagas disease (ChD) presented by dilated cardiomyopathy and heart failure. During infection, oxidative and nitrosative stresses are elicited by the immune cells for control the pathogen; however, excess nitric oxide and superoxide production can result in cysteine S-nitrosylation (SNO) of host proteins that affects cellular homeostasis and may contribute to disease development. To identify the proteins with changes in SNO modification levels as a hallmark of ChD, we obtained peripheral blood mononuclear cells (PBMC) from seronegative, normal healthy (NH, n = 30) subjects, and from seropositive clinically asymptomatic (ChD CA, n = 25) or clinically symptomatic (ChD CS, n = 28) ChD patients. All samples were treated (Asc+) or not-treated (Asc−) with ascorbate (reduces nitrosylated thiols), labeled with the thiol-labeling BODIPY FL-maleimide dye, resolved by two-dimensional electrophoresis (total 166 gels), and the protein spots that yielded significant differences in abundance or SNO level at p-value of ≤ 0.05t−test/Welch/BH were identified by MALDI-TOF/TOF MS or OrbiTrap LC-MS/MS. Targeted analysis of a new cohort of PBMC samples (n = 10–14/group) was conducted to verify the differential abundance/SNO levels of two of the proteins in ChD (vs. NH) subjects. The multivariate adaptive regression splines (MARS) modeling, comparing differences in relative SNO level (Asc−/Asc+ ratio) of the protein spots between any two groups yielded SNO biomarkers that exhibited ≥90% prediction success in classifying ChD CA (582-KRT1 and 884-TPM3) and ChD CS (426-PNP, 582-KRT1, 486-ALB, 662-ACTB) patients from NH controls. Ingenuity Pathway Analysis (IPA) of the SNO proteome dataset normalized to changes in protein abundance suggested the proteins belonging to the signaling networks of cell death and the recruitment and migration of immune cells were most affected in ChD CA and ChD CS (vs. NH) subjects. We propose that SNO modification of the select panel of proteins identified in this study have the potential to identify ChD severity in seropositive individuals exposed to Tc infection.
Highlights
Chagas disease (ChD), a neglected parasitic disease recognized as one of the top public health concern in the world, is endemic in Latin America and Mexico (Bonney, 2014)
Each peripheral blood mononuclear cells (PBMC) lysate was divided into two aliquots; aliquot A being reduced with ascorbate (Asc+, makes all cysteine residues available for BD labeling) and aliquot B (Asc−) was treated with neocuproine to preserve the SNO-modified cysteines
The protein spot intensities on BD-labeled Asc+ and Asc− normal healthy (NH), ChD clinically asymptomatic (CA), and ChD clinically symptomatic (CS) gels were normalized against the reference gel, and the data were analyzed in pair-wise manner
Summary
Chagas disease (ChD), a neglected parasitic disease recognized as one of the top public health concern in the world, is endemic in Latin America and Mexico (Bonney, 2014). With an increase in migratory movements, additional millions of people are at risk of infection in Latin America, Mexico, and southern regions of the United States, and in many countries of Europe where three million of migrants from American endemic areas are living (Bern et al, 2011; Tanowitz et al, 2016a; Monge-Maillo and Lopez-Velez, 2017). An asymptomatic phase occurs indeterminately, ∼30% of the infected people could develop chronic Chagas disease (ChD) that results in cardiomyopathy and heart failure (Machado et al, 2012; Bonney et al, 2019). T. cruzi infection is treated with benznidazole or nifurtimox. These drugs are effective in children presenting the acute infection phase (Perez-Molina and Molina, 2018), but exhibit limited efficacy and high toxicity in infected adults that are at risk of developing heart failure (Viotti et al, 2014). The current methods of detecting infection is by microscopic examination of blood smears, serology, or PCR (Ribeiro et al, 2012), but no methods currently exist to track or predict ChD progression (Balouz et al, 2017)
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