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

Michaela Procházková,Brian Panicucci,Alena Zíková

doi:10.1038/s41598-018-23472-6

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.

Highlights

Are Trypanosoma brucei medically and economically important parasites that cause disease in humans and livestock, but these flagellated protists are an excellent model to address fundamental questions within eukaryotic cell biology[1]
The generation of a viable double knockout TbMrf[1] cell line indicates that this release factor is not essential for the reduced mitochondrion of bloodstream form (BF) T. brucei
While the double knockout (dKO) TbMrf1 1wk cell line demonstrates the largest phenotypic changes compared to BF 427, we have demonstrated that the severity of these measured outcomes reduces over time as the cells are maintained in culture

Summary

Introduction

Transcription of the maxicircle DNA generates polycistronic precursors that are processed into two ribosomal RNAs and 18 mRNAs that encode subunits of the mitoribosome and the oxidative phosphorylation complexes. It is proposed that RPS12 acts as a functional link between RNA editing and translation in T. brucei[7] Due to their complex mt gene expression, several hundreds of proteins are required to synthesize the only two mt encoded proteins (RPS12 and FoF1-ATPase subunit A6) deemed to be essential for the extracellular pathogen to reside in the bloodstream of its mammalian host[7,21]. While the hydrolytic function of this enzyme is still essential, there is a greater reliance on the ADP/ATP carrier (TbAAC)[27,28] to provide ATP substrate, but to maintain the ∆ψm through the electrogenic exchange of cytosolic ATP4− for mt ADP3−21,29

Methods

Results

Conclusion