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

Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a homologue of the translocase of the mitochondria! inner membrane 50 (TbTim50). It has a pair of characteristic phosphatase signature motifs, DXDX(TN). Here, we demonstrated that, besides its protein phosphatase activity, the recombinant TbTim50 binds and hydrolyzes phosphatidic acid in a concentration-dependent manner. Mutations of D-242 and D-244, but not of D-345 and D-347, to alanine abolished these activities. In silico structural homology models identified the putative binding interfaces that may accommodate different phosphosubstrates. 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 G(2)/M phase and increased that of G, phase cells; inhibited segregation and caused overreplication of kinetoplast DNA (kDNA), and reduced BF cell growth. Depletion of TbTim50 increased the levels of AMPK phosphorylation, and parasite morphology was changed with upregulation of expression of a few stumpy marker genes. Importantly, we observed that TbTim50-depleted parasites were unable to establish infection in mice. Proteomics analysis showed reductions in levels of the translation factors, flagellar transport proteins, and many proteasomal subunits, including those of the mitochondria! heat shock locus ATPase (HsIVU), which is known to play a role in regulation of kinetoplast DNA (kDNA) replication. Reduction of the level of HsIV in TbTim50 KD cells was further validated by immunoblot analysis. Together, our results showed that TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and the cell cycle in the BF. Therefore, TbTim50 is an important target for structure-based drug design to combat African trypanosomiasis. IMPORTANCE African trypanosomiasis is a neglected tropical disease caused by the parasitic protozoan Trypanosoma brucei. During its digenetic life cycle, T. brucei undergoes multiple developmental changes to adapt in different environments. T. brucei BF parasites, dwelling in mammalian blood, produce ATP from glycolysis and hydrolyze ATP in mitochondria for generation of inner membrane potential. We found that TbTim50, a haloacid dehalogenase (HAD) family phosphatase, is critical for T. brucei BF survival in vitro and in vivo. Depletion of TbTim50 in BF reduced levels of CL and mitochondria Delta Psi and caused a detrimental effect on many cellular functions. Cells accumulated in the G, phase, and the kinetoplast was overreplicated, likely due to depletion of mitochondrial proteasome (mitochondria) heat shock locus ATPase [HsIVU]), a master regulator of kDNA replication. Cell growth inhibition was accompanied by changes in morphology, AMPK phosphorylation, and upregulation of expression of a few stumpy-specific genes. TbTim50 is essential for T. brucei survival and is an important therapeutic target for African trypanosomiasis.

Automated summary

TbTim50 is essential for mitochondrial function, regulation of kDNA replication, and the cell cycle in the bloodstream form of T. brucei. Depletion of TbTim50 results in inhibited cell growth, changes in morphology, increased AMPK phosphorylation, and upregulation of stumpy marker genes. TbTim50 is a crucial therapeutic target for African trypanosomiasis.