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Poster
33 |
Pharmacological inhibition of the vacuolar ATPase in bloodstream form Trypanosoma brucei rescues genetic knockdown of mitochondrial gene expression |
The survival of Trypanosoma brucei depends on maintenance and function of the mitochondrial genome (kinetoplast, or kDNA). However, a single point mutation in subunit γ of the mitochondrial F1FO ATPase can abolish the requirement of kDNA for survival of the bloodstream form of the parasite (Dean et al. 2013), and it was found that genetic or chemical inhibition of the vacuolar ATPase (V-ATPase) could have a similar effect (Baker et al. 2015). Here we present detailed studies of the effects of V-ATPase inhibitors on parasite growth after genetic or chemical interference with kDNA function. We found that treating bloodstream form T. brucei cells with bafilomycin A1 fully rescued loss of viability induced by depletion of the essential RNA editing ligase REL1 via genetic repression (‘conditional knock-out’; (Schnaufer et al. 2001). This confirmed a connection between V-ATPase function and kinetoplast dependency and suggests that V-ATPase inhibitors could be used as chemical tools in the study of kinetoplast biology. We also confirmed attenuation of the growth defect caused by isometamidium concentrations known to induce kDNA loss (Baker et al. 2015), although the rescue was only partial and not equivalent to the compensatory effect afforded be subunit γ mutations. Surprisingly, this rescue was not robustly replicated when using the chemically related compound ethidium bromide. In addition, bafilomycin A1 could not rescue from kDNA loss induced by RNAi-mediated knock-down of essential proteins PNT1 or TAC102 (Grewal et al. 2016; Trikin et al. 2016). Work is ongoing to investigate these unexpected findings in more detail.
Baker, N., G. Hamilton, J. M. Wilkes, S. Hutchinson, M. P. Barrett, and D. Horn. 2015. 'Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes', Proc Natl Acad Sci U S A, 112: 9112-7.
Dean, Samuel, Matthew K. Gould, Caroline E. Dewar, and Achim C. Schnaufer. 2013. 'Single point mutations in ATP synthase compensate for mitochondrial genome loss in trypanosomes', Proceedings of the National Academy of Sciences, 110: 14741-46.
Grewal, Jaspreet S., Karen McLuskey, Debanu Das, Elmarie Myburgh, Jonathan Wilkes, Elaine Brown, Leandro Lemgruber, Matthew K. Gould, Richard J. Burchmore, Graham H. Coombs, Achim Schnaufer, and Jeremy C. Mottram. 2016. 'PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast', The Journal of Biological Chemistry, 291: 9492-500.
Schnaufer, Achim, Aswini K. Panigrahi, Brian Panicucci, Robert P. Igo, Reza Salavati, and Kenneth Stuart. 2001. 'An RNA Ligase Essential for RNA Editing and Survival of the Bloodstream Form of Trypanosoma brucei', Science, 291: 2159-62.
Trikin, Roman, Nicholas Doiron, Anneliese Hoffmann, Beat Haenni, Martin Jakob, Achim Schnaufer, Bernd Schimanski, Benoît Zuber, and Torsten Ochsenreiter. 2016. 'TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes', PLOS Pathogens, 12: e1005586.
