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      Comparative SILAC Proteomic Analysis of Trypanosoma brucei Bloodstream and Procyclic Lifecycle Stages

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      Journal: PLoS ONE
      Publisher: Public Library of Science

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          Abstract

          The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level.

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          Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.

          Shao-En Ong, Blagoy Blagoev, Irina Kratchmarova … (2002)
          Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of specific amino acids into all mammalian proteins. Mammalian cell lines are grown in media lacking a standard essential amino acid but supplemented with a non-radioactive, isotopically labeled form of that amino acid, in this case deuterated leucine (Leu-d3). We find that growth of cells maintained in these media is no different from growth in normal media as evidenced by cell morphology, doubling time, and ability to differentiate. Complete incorporation of Leu-d3 occurred after five doublings in the cell lines and proteins studied. Protein populations from experimental and control samples are mixed directly after harvesting, and mass spectrometric identification is straightforward as every leucine-containing peptide incorporates either all normal leucine or all Leu-d3. We have applied this technique to the relative quantitation of changes in protein expression during the process of muscle cell differentiation. Proteins that were found to be up-regulated during this process include glyceraldehyde-3-phosphate dehydrogenase, fibronectin, and pyruvate kinase M2. SILAC is a simple, inexpensive, and accurate procedure that can be used as a quantitative proteomic approach in any cell culture system.
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            A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei.

            E. Wirtz, S. Leal, C Ochatt … (1999)
            First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.
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              Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication.

              R Brun, Schönenberger (1979)
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                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2012
                Publication date (Electronic): 4 May 2012
                Volume: 7
                Issue: 5
                Electronic Location Identifier: e36619
                Affiliations
                [1]Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Dundee, United Kingdom
                University of Texas-Houston Medical School, United States of America
                Author notes

                Conceived and designed the experiments: MDU MLSG MAJF. Performed the experiments: MDU MLSG. Analyzed the data: MDU. Wrote the paper: MDU.

                Article
                Publisher ID: PONE-D-12-03861
                DOI: 10.1371/journal.pone.0036619
                PMC ID: 3344917
                PubMed ID: 22574199
                SO-VID: 1eb7d5fa-de5a-4a3f-bb43-574efdc52b14
                Copyright © Urbaniak et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 7 February 2012
                Date accepted : 3 April 2012
                Page count
                Pages: 8
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Genetics
                Subject: Gene Expression
                Subject: Protein Translation
                Subject: Genomics
                Subject: Functional Genomics
                Subject: Genome Expression Analysis
                Subject: Microbiology
                Subject: Parasitology
                Subject: Quantitative Parasitology
                Subject: Protozoology
                Subject: Parastic Protozoans
                Subject: Trypanosoma
                Subject: Proteomics
                Subject: Protein Abundance

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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