Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry

Abstract

BackgroundTrypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology.ResultsUsing Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite.ConclusionsWe can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particularly of mitochondrial proteins in these cells when compared to the pleomorphic in vivo situation. In order to better understand the different levels of gene expression regulation in this organism we compared mRNA steady state abundance with the relative protein abundance-changes and detected moderate but significant correlation indicating that trypanosomes possess a significant repertoire of translational and posttranslational mechanisms to regulate protein abundance.

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

“…Indeed, in a comparative SILAC study, the putG3PDH protein was detected 5 times more often in PF than in BF (44), and yet two other studies using the same protein-labeling method failed to detect putG3PDH at all (45, 46) (see Table S4 in the supplemental material). Although this discrepancy might reflect different proteomics approaches, one cannot exclude the possibility that it has been influenced by the different cultivation media and strains used (44)(45)(46).…”

“…In contrast to PF, the BF cells do not contain a succinate-producing branch in their glycosomes (9) and hence exclusively regenerate the reduced cofactors required for glycolysis via the G3P:DHAP shuttle using mtG3PDH (13,14). The importance of mtG3PDH for the BF cells is further reflected by its upregulation compared to PF in this life stage at both the RNA level (42,43) and the protein level (44)(45)(46). The essentiality of mtG3PDH for BF is therefore not surprising, while depletion of the same protein in PF, reflected in the absence of its respective enzymatic activity, did not result in an altered growth phenotype, regardless of whether regular or lowglucose SDM-79 medium was used (data not shown).…”

A component of the mitochondrial outer membrane proteome of T. brucei probably contains covalent bound fatty acids

“…POMP39 has neither a predicted transmembrane helix nor a signal anchor. The polypeptide is strongly overexpressed in the BSF, as evident from proteomics studies (Bridges et al, 2008;Gunasekera et al, 2012).…”

“…POMP39 is strongly upregulated in the disease-causing BSF of the parasite (Gunasekera et al, 2012;Urbaniak et al, 2012). Consequently, the subcellular localization of POMP39 was also investigated in the BSF.…”

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei

“…To enable infection of tsetse, the proliferating long slender bloodstream forms first need to differentiate to cell-cycle arrested short stumpy forms [1]. These express a specific set of genes pre-adapting them for transfer to the tsetse fly, notably genes associated with mitochondrial metabolism [2][3][4][5][6]. When the fly ingests a blood meal, stumpy forms differentiate to procyclic forms (PF) in the lumen of the midgut [7].…”