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

Abstract

AbstractThe 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 thePEPCKgene knockout (Δpepck) leads to the selection of such a deletion event between theFRDgandFRDm2genes to produce a chimericFRDg-m2gene in the Δpepck*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 Δpepck*cells, while inhibition ofFRDg-m2expression had no effect, which indicated that this recombination event has been selected in the Δpepck*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.