PMID- 34517757
OWN - NLM
STAT- MEDLINE
DCOM- 20220207
LR  - 20220207
IS  - 2150-7511 (Electronic)
VI  - 12
IP  - 5
DP  - 2021 Oct 26
TI  - Trypanosoma brucei Tim50 Possesses PAP Activity and Plays a Critical Role in Cell 
      Cycle Regulation and Parasite Infectivity.
PG  - e0159221
LID - 10.1128/mBio.01592-21 [doi]
LID - e01592-21
AB  - Trypanosoma brucei, the infective agent for African trypanosomiasis, possesses a 
      homologue of the translocase of the mitochondrial inner membrane 50 (TbTim50). It 
      has a pair of characteristic phosphatase signature motifs, DXDX(T/V). 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 
      (DeltaPsi). TbTim50 knockdown (KD) also reduced the population of G(2)/M phase and 
      increased that of G(1) 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 mitochondrial heat shock locus ATPase (HslVU), which is 
      known to play a role in regulation of kinetoplast DNA (kDNA) replication. 
      Reduction of the level of HslV 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 mitochondrial DeltaPsi and caused a 
      detrimental effect on many cellular functions. Cells accumulated in the G(1) 
      phase, and the kinetoplast was overreplicated, likely due to depletion of 
      mitochondrial proteasome (mitochondrial heat shock locus ATPase [HslVU]), 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.
FAU - Tripathi, Anuj
AU  - Tripathi A
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Singha, Ujjal K
AU  - Singha UK
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Cooley, Ayorinde
AU  - Cooley A
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Gillyard, Taneisha
AU  - Gillyard T
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Krystofiak, Evan
AU  - Krystofiak E
AD  - Department of Cell and Developmental Biology, Vanderbilt University, Nashville, 
      Tennessee, USA.
FAU - Pratap, Siddharth
AU  - Pratap S
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Davis, Jamaine
AU  - Davis J
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
FAU - Chaudhuri, Minu
AU  - Chaudhuri M
AUID- ORCID: 0000-0001-7626-1280
AD  - Department of Microbiology, Immunology, and Physiology, School of Medicine, 
      Meharry Medical Collegegrid.259870.1, Nashville, Tennessee, USA.
LA  - eng
GR  - R01 AI125662/AI/NIAID NIH HHS/United States
GR  - P30 DK058404/DK/NIDDK NIH HHS/United States
GR  - T32 AI007281/AI/NIAID NIH HHS/United States
GR  - U54 RR026140/RR/NCRR NIH HHS/United States
GR  - S21 MD000104/MD/NIMHD NIH HHS/United States
GR  - P30 CA068485/CA/NCI NIH HHS/United States
GR  - U54 MD007593/MD/NIMHD NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20210914
PL  - United States
TA  - mBio
JT  - mBio
JID - 101519231
RN  - 0 (DNA, Kinetoplast)
RN  - 0 (Protozoan Proteins)
SB  - IM
MH  - Animals
MH  - *Cell Cycle
MH  - Cell Line
MH  - DNA, Kinetoplast/genetics/metabolism
MH  - Mice
MH  - Mice, Inbred BALB C
MH  - Mitochondria/metabolism
MH  - Phosphorylation
MH  - Protozoan Proteins/genetics
MH  - Trypanosoma brucei brucei/*genetics/*metabolism
MH  - Trypanosomiasis, African/*parasitology
PMC - PMC8546626
OTO - NOTNLM
OT  - Trypanosoma brucei
OT  - cell cycle
OT  - infectivity
OT  - kDNA
OT  - mitochondria
OT  - protein translocase
EDAT- 2021/09/15 06:00
MHDA- 2022/02/08 06:00
PMCR- 2021/09/14
CRDT- 2021/09/14 05:37
PHST- 2021/09/15 06:00 [pubmed]
PHST- 2022/02/08 06:00 [medline]
PHST- 2021/09/14 05:37 [entrez]
PHST- 2021/09/14 00:00 [pmc-release]
AID - mBio01592-21 [pii]
AID - mbio.01592-21 [pii]
AID - 10.1128/mBio.01592-21 [doi]
PST - ppublish
SO  - mBio. 2021 Oct 26;12(5):e0159221. doi: 10.1128/mBio.01592-21. Epub 2021 Sep 14.