Dataset Information

TcI and TcII DTUs isolated from chronic chagasic cardiac patients LC-MSMS

ABSTRACT: This work aimed to investigate the differential expression of proteins from two distinct genetic groups of T. cruzi (´DTUs´, TcI and TcII), isolated from chronically infected individuals displaying the cardiac form of Chagas’ disease. For this purpose, epimastigote forms of the two isolates were cultured in vitro and the cells recovered for protein extraction. Comparative two-dimensional gel electrophoreses were performed and differentially expressed spots selected for identification by mass spectrometry, followed by database searching and protein categorization. The two-dimensional electrophoretic profiles revealed the complex composition of the T. cruzi extracted proteome. In total 46 differentially expressed proteins were identified present in 40 distinct spots found in the comparative gel analyses. The comparative two-dimensional gel electrophoresis allowed detection of major differences in protein expression between two T. cruzi isolates, belonging to the TcI and TcII genotypes. Our findings suggest that patients displaying the cardiac form of the disease harbor parasites capable of exhibiting distinct proteomic profiles.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Trypanosoma Cruzi

TISSUE(S): Epimastigote

DISEASE(S): Trypanosomiasis

SUBMITTER: William Borges

LAB HEAD: William Castro Borges

PROVIDER: PXD011172 | Pride | 2019-11-12

REPOSITORIES: Pride

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Publications

Differential expression of proteins in genetically distinct Trypanosoma cruzi samples (TcI and TcII DTUs) isolated from chronic Chagas disease cardiac patients.

Tavares de Oliveira Maykon M Taciana Santos Silva Karina K Xavier Neves Leandro L de Ornelas Toledo Max Jean MJ Castro-Borges William W de Lana Marta M

Parasites & vectors 20181129 1

<h4>Background</h4>Trypanosoma cruzi, a hemoflagellate protozoan parasite and the etiological agent of Chagas disease (CD), exhibits great genetic and biological diversity. Infected individuals may present clinical manifestations with different levels of severity. Several hypotheses have been proposed to attempt to correlate the diversity of clinical signs and symptoms to the genetic variability of T. cruzi. This work aimed to investigate the differential expression of proteins from two distinct ...[more]

PMID: 30497493

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Project description:RNA pull-down assay.<br>For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified.<br><br>RNA purification and amplification:<br><br>RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual.<br><br>Microarray analysis:<br>The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. <br>Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

Dataset Information

TcI and TcII DTUs isolated from chronic chagasic cardiac patients LC-MSMS

Publications

Differential expression of proteins in genetically distinct Trypanosoma cruzi samples (TcI and TcII DTUs) isolated from chronic Chagas disease cardiac patients.

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