Cultured bloodstream Trypanosoma brucei adapt to life without mitochondrial translation release factor 1

Abstract

Trypanosoma brucei is an extracellular parasite that alternates between an insect vector (procyclic form) and the bloodstream of a mammalian host (bloodstream form). While it was previously reported that mitochondrial release factor 1 (TbMrf1) is essential in cultured procyclic form cells, we demonstrate here that in vitro bloodstream form cells can tolerate the elimination of TbMrf1. Therefore, we explored if this discrepancy is due to the unique bioenergetics of the parasite since procyclic form cells rely on oxidative phosphorylation; whereas bloodstream form cells utilize glycolysis for ATP production and FoF1-ATPase to maintain the essential mitochondrial membrane potential. The observed disruption of intact bloodstream form FoF1-ATPases serves as a proxy to indicate that the translation of its mitochondrially encoded subunit A6 is impaired without TbMrf1. While these null mutants have a decreased mitochondrial membrane potential, they have adapted by increasing their dependence on the electrogenic contributions of the ADP/ATP carrier to maintain the mitochondrial membrane potential above the minimum threshold required for T. brucei viability in vitro. However, this inefficient compensatory mechanism results in avirulent mutants in mice. Finally, the depletion of the codon-independent release factor TbPth4 in the TbMrf1 knockouts further exacerbates the characterized mitchondrial phenotypes.

Bioenergetic consequences of FoF1–ATP synthase/ATPase deficiency in two life cycle stages of Trypanosoma brucei

“…Nevertheless, while Tb1 and OSCP suppression down to 3 to 10% caused a corresponding loss of F o F 1 –ATPase complexes, these BSF trypanosomes were able to thrive in culture without any obvious effect on their viability. Similarly, perturbation of mitochondrial translation in BSF cells, manifested by decreased levels of F o F 1 –ATPase due to reduced production of its mitochondrial encoded subunit a, had no effect on growth in culture ( 44 ). Moreover, the ΔΨm of BSF Tb1 5’ and OSCP RNAi–induced cells was not affected when measured by flow cytometry of live cells, but there was a significant reduction in the ability of the F o F 1 –ATPase to generate a proton gradient when estimated in permeabilized cells by safranin O assay.…”

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation

“…Treatment with FCCP, a protonophore, induced membrane depolarization as expected ( Fig 2G ). To determine if the AAC DKO mutants still maintain their ΔΨ m by the reverse activity of F o F 1 -ATP synthase, the BSF 427 and AAC DKO cells were incubated for 24 hours with a sublethal concentration of the F o F 1 -ATP synthase inhibitor, oligomycin (250 ng/ml, ~0.5 of the EC 50 for BSF 427 [ 22 , 28 ]). While this treatment did not affect the doubling time of BSF 427 or AAC DKO parasites (BSF 427: 6 ± 0.2 hours, AAC DKO: 6.3 ± 0.3), it did result in a similar reduction of the ΔΨ m in BSF 427 and AAC DKO mutants, with values reaching 50±11% and 64±7%, respectively ( Fig 2H ).…”

“…Remarkably, the BSF parasites exhibit ~40-fold lower sensitivity to AAC inhibitors than dyskinetoplastic trypanosomes, which lack their mitochondrial genome and thus the proton-pumping F o F 1 -ATPase activity [ 28 ]. Even though these dyskinetoplastic trypanosomes fully rely on the electrogenic exchange of ATP 4- /ADP 3- to generate ΔΨ m [ 29 ], the striking difference in sensitivity to AAC inhibitors raises questions about the role of AAC for BSF mitochondria.…”

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active

“…PTMs have been described in trypanosomes [71] and are a freely reversible mechanism of control at the protein level that would enable the parasites to react quickly to environmental changes, including the presumably abrupt changes associated with vector (tsetse fly) to host transmission [72]. The metabolic change in procyclic trypanosomes from glycolysis to oxidative phosphorylation due to activation of the mitochondrion [73,74] could enable reactive oxygen species (ROS) to oxidize a Met residue to MetO [75]. The reversible formation of MetO by ROS as a mechanism of enzymatic activation has been described [76,77]; and the enzyme that mediates the reversal of this oxidation, methionine sulfoxide reductase, is putatively localized in the T. brucei genome (Tb927.8.550).…”