The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

Abstract

␣-Ketoglutarate decarboxylase (␣-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, ␣-KDE1 was expressed in BF T. brucei and RNA interference knockdown of ␣-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of ␣-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of ␣-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of ␣-KDE1 contain overlapping mitochondrion-and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that ␣-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.

Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia

“…Similar filamentous structures were also visible on T. brucei in the blood of infected mice (Figures 1B and S1A). The formation of the filaments in vitro was stimulated by stress from RNAi against an essential BF protein or by addition of complement active serum to growth medium (Figure S1B) (Sykes et al, 2015). These induction mechanisms allowed us to investigate filament biogenesis.…”

“…Production of nanotubes and EVs was stimulated by stressing BF T. brucei with RNAi against the essential BF protein α-KDE1 or by the addition of complement active FBS (Sykes et al, 2015). In all other experiments, EVs were acquired from trypanosomes grown in heat inactivated serum or serum free conditions (for mass spectrometry).…”

“…All DIC and immunofluorescence images were acquired with a Zeiss Axio Observer Z1 as previously described Sykes et al, 2015.…”

“…BF T. brucei stimulated for the formation of membrane nanotubes were fixed and images were acquired with a JEOL-JEM 1210 transmission electron microscope as previously described (Sykes et al, 2015). For SEM, 2.5% glutaraldehyde-fixed cells were dehydrated on a 0.22 mm membrane, critical point dried, sputter coated with gold and visualized with a Zeiss 1450EP scanning electron microscope.…”

“…Total cell and purified EV proteins were fractionated by reducing SDS-PAGE and western blotted as previously described using 5 × 10 5 BF trypanosomes or EVs purified from 2 × 10 6 cells (Sykes et al, 2015). …”