Dataset Information

Metabolic selection of a homologous recombination mediated gene rearrangement in Trypanosoma brucei

ABSTRACT: The Trypanosoma brucei genome contains three fumarate reductase (FRD) genes, including two tandemly arranged in chromosome 5 encoding the glycosomal isoform (FRDg) and a potentially mitochondrial FRD isoform never detected so far in trypanosomes (FRDm2). We report here that the PPDK/PEPCK/GPDH mutant cell line (Δppdk/Δpepck/RNAi-GPDH) expresses a chimeric non-functional FRDg-m2 isoform resulting from stochastic homologous recombination within the FRDg/FRDm2 locus. This conclusion come from comparison of the parental (WT, Δppdk, Δpepck and Δppdk/Δpepck) and Δppdk/Δpepck/RNAi-GPDH proteomes. The selective advantage provided by the consequential loss of the FRDg gene, in the context of the Δppdk/Δpepck/RNAi-GPDH null background, is probably related to the autoflavinylation activity of the FRDg isoform.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Dupuy Jean-William

LAB HEAD: Frédéric Bringaud

PROVIDER: PXD020185 | Pride | 2021-06-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	Linkbetweenfiles.xlsx	Xlsx
	Resultsofexperimentsbasedonglycosome-enrichedextracts.xlsx	Xlsx
	Resultsofexperimentsbasedontotalextracts.xlsx	Xlsx
	TriTrypDB-46_TbruceiTREU927_AnnotatedProteins.fasta	Fasta
	fb110330_S1_A12_01.RAW	Raw

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Publications

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

Wargnies Marion M Plazolles Nicolas N Schenk Robin R Villafraz Oriana O Dupuy Jean-William JW Biran Marc M Bachmaier Sabine S Baudouin Hélène H Clayton Christine C Boshart Michael M Bringaud Frédéric F

The Journal of biological chemistry 20210101

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase ( ...[more]

PMID: 33741344

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Project description:The bloodstream forms of Trypanosoma brucei (BSF), responsible for the sleeping sickness, primarily evaluate in the blood of its mammalian hosts. The skin and the adipose tissues were recently identified as additional major sites for the parasite proliferation and transmission through its insect vector blood ingestion. Glucose is the only carbon source used by the blood parasites to feed its central metabolism, however, the metabolic behavior of the tissue-adapted parasites has not yet been addressed. Since production of glycerol is an important primary function of adipocytes, we have adapted the BSF trypanosomes to glucose-depleted and glycerol-rich growth medium (CMM_Gly/GlcNAc) and compared its metabolism and proteome with standard glucose-rich growth conditions (CMM_Glu). BSF consume 2-times more oxygen per carbon consumed in CMM_Gly/GlcNAc and is 11.5-times more sensitive to SHAM, a specific inhibitor of the alternative oxidase, which is consistent with the expected absolute requirement of the mitochondrial respiratory chain activity to convert glycerol into dihydroxyacetone phosphate. Metabolomic analyses by mass spectrometry showed that 13C-labeled glycerol is incorporated into hexose phosphates through gluconeogenesis. After several weeks of growth in CMM_Gly/GlcNAc, BSF adapt their metabolism by increasing and decreasing the rate of glycerol and glucose consumption, respectively. As expected RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol degradation and is lethal for BSF growth in CMM_Gly/GlcNAc. However, the glycerol kinase activity is 7.8-fold decreased in CMM_Gly/GlcNAc, as confirms by western blotting and proteomic analyses, which suggests that the huge excess of glycerol kinase, which is not necessary for glycerol metabolism, is required for another yet undetermined non-essential function in glucose rich-conditions. Altogether these data shows that BSF trypanosomes are well adapted to glycerol-rich conditions, which could be encountered by the parasite in extravascular niches, such as skin and adipose tissues.