NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms

This work was supported by a grant from the National Institutes of Health (USA) [AI 5R01 AI069057] to MP and AS; a grant from the Medical Research Council (UK) [G0600129] to AS; grants from the Centre National de la Recherche Scientifique (CNRS, France), The Université de Bordeaux, The Agence Nationale de la Recherche (ANR) [ACETOTRYP of the ANR-BLANC-2010 call and GLYCONOV of the “Générique” call] and the Laboratoire d’Excellence (LabEx) ParaFrap [grant ANR-11-LABX-0024] to FB; and a grant from the Wellcome Trust [093228] to TKS.In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite’s second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion. While inducible knockdown of NDH2 had a modest growth effect in bloodstream forms, NDH2 null mutants, as well as inducible knockdowns in a complex I deficient background, showed a greater reduction in growth. Altering the NAD+/NADH balance would affect numerous processes directly and indirectly, including acetate production. Indeed, loss of NDH2 led to reduced levels of acetate, which is required for several essential pathways in bloodstream form T. brucei and which may have contributed to the observed growth defect. In conclusion our study shows that NDH2 is important, but not essential, in proliferating bloodstream forms of T. brucei, arguing that the mitochondrial NAD+/NADH balance is important in this stage, even though the mitochondrion itself is not actively engaged in the generation of ATP.Publisher PDFPeer reviewe

Enigmatic presence of mitochondrial complex I in Trypanosoma brucei bloodstream forms

The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into α and β subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740- and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance