Biology subjects

Boshart, M.

Publications and source records attributed to Boshart, M..

3 recordsLinked to original sources

bioRxiv2020

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei

3A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N-terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4 kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

biochemistry↗

bioRxiv2020

Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly, where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and -ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of a dozen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of -ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) -ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an -ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6, showed a growth defect in the presence of 2 mM proline, which is rescued by -ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly. Author SummaryIn the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. Here we show that tricarboxylic acid (TCA) cycle intermediates, i.e. succinate, malate and -ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. Some of these additional carbon sources are needed for the development of epimastigotes, which differentiate from procyclics in the midgut of the fly, since their growth defect observed in the presence of 2 mM proline is rescued by addition of -ketoglutarate. In addition, we have implemented new approaches to study a poorly explored branch of the TCA cycle converting malate to -ketoglutarate, which was previously described as non-functional in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasites metabolism in various organ systems of the tsetse fly, where it evolves.

microbiology↗

bioRxiv2020

Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei

The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout ({Delta}pepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the {Delta}pepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the {Delta}pepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the {Delta}pepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.

microbiology↗