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bioRxiv · 10.1101/2021.04.26.441502

Trypanosoma brucei Tim50 Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity

Tripathi, A.·Singha, U. K.·Cooley, A.·Gillyard, T.·Krystofiak, E.·Pratap, S.·Davis, J.·Chaudhuri, M.
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Abstract

Tim50 is a receptor subunit of the preprotein-translocase of the mitochondrial inner membrane, TIM23. Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of Tim50 (TbTim50) with a pair of characteristic DXDX(T/V) phosphatase signature motifs. Here, we demonstrated that besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. In silico structural homology models identify the putative binding interfaces that may accommodate different phospho-substrates. Interestingly, TbTim50 depletion in the bloodstream form (BF) of T. brucei reduced cardiolipin (CL) levels and decreased mitochondrial membrane potential ({Delta}{Psi}). TbTim50 knockdown (KD) also reduced the population of G2 phase and increased G1 phase; thus, BF cell growth was reduced. Confocal and electron microscopy revealed a defect in regulation of kinetoplast (kDNA) replication due to TbTim50 KD. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed to stumpy-like with upregulation of few stumpy marker gene expressions. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice and rats. Proteomics analysis showed reductions of the translation factors, flagellar transport proteins, and many proteasomal subunits, including the mitochondrial HslVU that is known to play a role in kDNA replication. Reduction of the level of HslV in TbTim50 KD cells was further validated by immunoblot analysis. Altogether, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. ImportanceAfrican trypanosomiasis, a neglected tropical disease caused by parasitic protozoan Trypanosoma brucei, is transmitted by the tsetse fly prevalent in sub-Saharan Africa. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF, dwelling in mammalian blood, generates ATP from glycolysis and hydrolyzes ATP in mitochondria for inner membrane potential. We found that TbTim50, a HAD-family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced CL levels and mitochondrial {Delta}{Psi} and caused a detrimental effect on many cellular functions. Cells accumulated in G1-S phase, and kinetoplast was over-replicated due to depletion of mitochondrial proteasomes, HslVU, a master-regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of stumpy-specific gene expression. TbTim50 is essential for T. brucei survival and an important T. brucei therapeutic target.

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Tripathi, A., Singha, U. K., Cooley, A., Gillyard, T., Krystofiak, E., Pratap, S., Davis, J., Chaudhuri, M.. 2021-04-27. Trypanosoma brucei Tim50 Plays a Critical Role in Cell Cycle Regulation and Parasite Infectivity. https://doi.org/10.1101/2021.04.26.441502

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