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J Biol Chem. 2016 Apr 29;291(18):9492-500. doi: 10.1074/jbc.M116.714972. Epub 2016 Mar 3.

PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.

Author information

1
From the Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom, the Department of Biology, Centre for Immunology and Infection, University of York, Wentworth Way, Heslington, York YO10 5DD, United Kingdom.
2
From the Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom.
3
the Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
4
the Institute of Immunology and Infection Research and Centre for Immunity, Infection, and Evolution, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom.
5
the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom.
6
From the Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, United Kingdom, the Department of Biology, Centre for Immunology and Infection, University of York, Wentworth Way, Heslington, York YO10 5DD, United Kingdom, jeremy.mottram@york.ac.uk.

Abstract

The structure of a C11 peptidase PmC11 from the gut bacterium, Parabacteroides merdae, has recently been determined, enabling the identification and characterization of a C11 orthologue, PNT1, in the parasitic protozoon Trypanosoma brucei. A phylogenetic analysis identified PmC11 orthologues in bacteria, archaea, Chromerids, Coccidia, and Kinetoplastida, the latter being the most divergent. A primary sequence alignment of PNT1 with clostripain and PmC11 revealed the position of the characteristic His-Cys catalytic dyad (His(99) and Cys(136)), and an Asp (Asp(134)) in the potential S1 binding site. Immunofluorescence and cryoelectron microscopy revealed that PNT1 localizes to the kinetoplast, an organelle containing the mitochondrial genome of the parasite (kDNA), with an accumulation of the protein at or near the antipodal sites. Depletion of PNT1 by RNAi in the T. brucei bloodstream form was lethal both in in vitro culture and in vivo in mice and the induced population accumulated cells lacking a kinetoplast. In contrast, overexpression of PNT1 led to cells having mislocated kinetoplasts. RNAi depletion of PNT1 in a kDNA independent cell line resulted in kinetoplast loss but was viable, indicating that PNT1 is required exclusively for kinetoplast maintenance. Expression of a recoded wild-type PNT1 allele, but not of an active site mutant restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. These data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.

KEYWORDS:

RNA interference (RNAi); Trypanosoma brucei; microscopy; parasite; peptidase

PMID:
26940875
PMCID:
PMC4850289
DOI:
10.1074/jbc.M116.714972
[Indexed for MEDLINE]
Free PMC Article

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