title: PNT1 Is a C11 Cysteine Peptidase Essential for Replication of the Trypanosome Kinetoplast.
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.
authors.affiliation: 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.
authors.forename: Jaspreet S
authors.identifier: http://orcid.org/0000-0002-2219-7655
authors.initials: JS
authors.lastname: Grewal
authors.affiliation: 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.
authors.forename: Karen
authors.identifier: http://orcid.org/0000-0002-9841-4943
authors.initials: K
authors.lastname: McLuskey
authors.affiliation: the Joint Center for Structural Genomics, Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, Menlo Park, California 94025.
authors.forename: Debanu
authors.initials: D
authors.lastname: Das
authors.affiliation: 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.
authors.forename: Elmarie
authors.initials: E
authors.lastname: Myburgh
authors.affiliation: 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.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Wilkes
authors.affiliation: 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.
authors.forename: Elaine
authors.initials: E
authors.lastname: Brown
authors.affiliation: 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.
authors.forename: Leandro
authors.initials: L
authors.lastname: Lemgruber
authors.affiliation: the Institute of Immunology and Infection Research and Centre for Immunity, Infection, and Evolution, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Gould
authors.affiliation: 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.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Burchmore
authors.affiliation: the Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, United Kingdom.
authors.forename: Graham H
authors.initials: GH
authors.lastname: Coombs
authors.affiliation: the Institute of Immunology and Infection Research and Centre for Immunity, Infection, and Evolution, University of Edinburgh, Edinburgh EH9 3FL, United Kingdom.
authors.forename: Achim
authors.initials: A
authors.lastname: Schnaufer
authors.affiliation: 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.
authors.forename: Jeremy C
authors.identifier: http://orcid.org/0000-0001-5574-3766
authors.initials: JC
authors.lastname: Mottram
mesh_terms: D000483:Alleles; D000818:Animals; D020134:Catalytic Domain; D057056:Cysteine Proteases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D051379:Mice; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics
chemical_list: D015800:Protozoan Proteins; D057056:Cysteine Proteases
keywords: RNA interference (RNAi); Trypanosoma brucei; microscopy; parasite; peptidase