Comprehensive Proteomic Analysis of <i>Trypanosoma cruzi</i> Epimastigote Cell Surface Proteins by Two Complementary Methods

Queiroz, Rayner M L; Charneau, Sébastien; Motta, Flávia Nader; Santana, Jaime M.; Roepstorff, Peter; Ricart, Carlos André Ornelas

doi:10.1021/pr400110h

Cited by 21 publications

(19 citation statements)

References 45 publications

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“…Recently, two comprehensive proteomic surveys of T. cruzi plasma membrane proteins have been reported (56,57). We observed that most of the 212 proteins regulated during amastigogenesis with predicted transmembrane domains were also detected in the reported plasma membrane subproteome of trypomastigotes and axenic amastigotes.…”

Section: Resultsmentioning

confidence: 72%

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Queiroz

Charneau

Mandacaru

et al. 2014

Molecular & Cellular Proteomics

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Chagas disease is a tropical neglected disease endemic in Latin America caused by the protozoan Trypanosoma cruzi. The parasite has four major life stages: epimastigote, metacyclic trypomastigote, bloodstream trypomastigote, and amastigote. The differentiation from infective trypomastigotes into replicative amastigotes, called amastigogenesis, takes place in vivo inside mammalian host cells after a period of incubation in an acidic phagolysosome. This differentiation process can be mimicked in vitro by incubating tissue-culture-derived trypomastigotes in acidic DMEM. Here we used this well-established differentiation protocol to perform a comprehensive quantitative proteomic and phosphoproteomic analysis of T. cruzi amastigogenesis. Samples from fully differentiated forms and two biologically relevant intermediate time points were Lys-C/trypsin digested, iTRAQ-labeled, and multiplexed. Subsequently, phosphopeptides were enriched using a TiO 2 matrix. Non-phosphorylated peptides were fractionated via hydrophilic interaction liquid chromatography prior to LC-MS/MS analysis. LC-MS/MS and bioinformatics procedures were used for protein and phosphopeptide quantitation, identification, and phosphorylation site assignment. We were able to identify regulated proteins and pathways involved in coordinating amastigogenesis. We also observed that a significant proportion of the regulated proteins were membrane proteins. Modulated phosphorylation events coordinated by protein kinases and phosphatases that are part of the signaling cascade induced by incubation in acidic medium were also evinced. To our knowledge, this work is the most comprehensive quantitative proteomics study of T. cruzi amastigogenesis, and these data will serve as a trustworthy basis for future studies, and possibly for new potential drug targets. Molecular & Cellular Proteomics 13:

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Section: Resultsmentioning

confidence: 72%

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Queiroz

Charneau

Mandacaru

et al. 2014

Molecular & Cellular Proteomics

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“…The number of proteins, protein groups, and number of peptides were filtered for False Discovery Rates (FDR) less than 1%. Only peptides with rank 1 and minimal of 2 peptides per protein were accepted for identification using Proteome Discoverer (Charneau et al, 2007 ; Queiroz et al, 2013 ).…”

Section: Methodsmentioning

confidence: 99%

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

et al. 2016

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Giardiasis, currently considered a neglected disease, is caused by the intestinal protozoan parasite Giardia duodenalis and is widely spread in human as well as domestic and wild animals. The lack of appropriate medications and the spread of resistant parasite strains urgently call for the development of novel therapeutic strategies. Host microbiota or certain probiotic strains have the capacity to provide some protection against giardiasis. By combining biological and biochemical approaches, we have been able to decipher a molecular mechanism used by the probiotic strain Lactobacillus johnsonii La1 to prevent Giardia growth in vitro. We provide evidence that the supernatant of this strain contains active principle(s) not directly toxic to Giardia but able to convert non-toxic components of bile into components highly toxic to Giardia. By using bile acid profiling, these components were identified as deconjugated bile-salts. A bacterial bile-salt-hydrolase of commercial origin was able to mimic the properties of the supernatant. Mass spectrometric analysis of the bacterial supernatant identified two of the three bile-salt-hydrolases encoded in the genome of this probiotic strain. These observations document a possible mechanism by which L. johnsonii La1, by secreting, or releasing BSH-like activity(ies) in the vicinity of replicating Giardia in an environment where bile is present and abundant, can fight this parasite. This discovery has both fundamental and applied outcomes to fight giardiasis, based on local delivery of deconjugated bile salts, enzyme deconjugation of bile components, or natural or recombinant probiotic strains that secrete or release such deconjugating activities in a compartment where both bile salts and Giardia are present.

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“…Previous proteomic studies have been carried out aiming the understanding of T . cruzi biology, including global proteomic analysis [ 7 , 8 ], proteomic changes during cell differentiation [ 9 , 10 ] and analysis of subproteomes such as secretome [ 11 ], cell surface [ 12 , 13 ], and organelles [ 14 ]. Here, we present for the first time, to the best of our knowledge, the isolation and proteome analysis of T .…”

Section: Introductionmentioning

confidence: 99%

Unveiling the Trypanosoma cruzi Nuclear Proteome

et al. 2015

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Replication of Trypanosoma cruzi, the etiological agent of Chagas disease, displays peculiar features, such as absence of chromosome condensation and closed mitosis. Although previous proteome and subproteome analyses of T. cruzi have been carried out, the nuclear subproteome of this protozoan has not been described. Here, we report, for the first time to the best of our knowledge, the isolation and proteome analysis of T. cruzi nuclear fraction. For that, T. cruzi epimastigote cells were lysed and subjected to cell fractionation using two steps of sucrose density gradient centrifugation. The purity of the nuclear fraction was confirmed by phase contrast and fluorescence microscopy. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) allowed the identification of 864 proteins. Among those, 272 proteins were annotated as putative uncharacterized, and 275 had not been previously reported on global T. cruzi proteome analysis. Additionally, to support our enrichment method, bioinformatics analysis in DAVID was carried out. It grouped the nuclear proteins in 65 gene clusters, wherein the clusters with the highest enrichment scores harbor members with chromatin organization and DNA binding functions.

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Comprehensive Proteomic Analysis of Trypanosoma cruzi Epimastigote Cell Surface Proteins by Two Complementary Methods

Cited by 21 publications

References 45 publications

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Quantitative Proteomic and Phosphoproteomic Analysis of Trypanosoma cruzi Amastigogenesis

Deconjugated Bile Salts Produced by Extracellular Bile-Salt Hydrolase-Like Activities from the Probiotic Lactobacillus johnsonii La1 Inhibit Giardia duodenalis In vitro Growth

Unveiling the Trypanosoma cruzi Nuclear Proteome

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