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 (His⁹⁹ and Cys¹³⁶), and an Asp (Asp¹³⁴) 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 mutantmore » restored parasite viability after induction in vitro and in vivo confirming that the peptidase activity of PNT1 is essential for parasite survival. Furthermore, these data provide evidence that PNT1 is a cysteine peptidase that is required exclusively for maintenance of the trypanosome kinetoplast.« less

Authors:

Grewal, Jaspreet S.  ^[1];

• Search DOE PAGES for author "Grewal, Jaspreet S."
• Search DOE PAGES for ORCID "0000-0002-2219-7655"

McLuskey, Karen  ^[2];

• Search DOE PAGES for author "McLuskey, Karen"
• Search DOE PAGES for ORCID "0000-0002-9841-4943"

Das, Debanu ^[3]; Myburgh, Elmarie ^[1]; Wilkes, Jonathan ^[2]; Brown, Elaine ^[1]; Lemgruber, Leandro ^[2]; Gould, Matthew K. ^[4]; Burchmore, Richard J. ^[2]; Coombs, Graham H. ^[5]; Schnaufer, Achim ^[4]; Mottram, Jeremy C.  ^[1]

• Search DOE PAGES for author "Mottram, Jeremy C."
• Search DOE PAGES for ORCID "0000-0001-5574-3766"

---

+ Show Author Affiliations

1. Univ. of Glasgow, Glasgow (United Kingdom); Univ. of York, York (United Kingdom)
2. Univ. of Glasgow, Glasgow (United Kingdom)
3. SLAC National Accelerator Lab., Menlo Park, CA (United States)
4. Univ. of Edinburgh, Edinburgh (United Kingdom)
5. Univ. of Strathclyde, Glasgow (United Kingdom)

Publication Date:

Thu Mar 03 00:00:00 EST 2016

Research Org.:

SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1256324

Grant/Contract Number:  

MR/K019384; 091790; 104111; U54 GM094586; AC02-76SF00515

Resource Type:

Accepted Manuscript

Journal Name:

Journal of Biological Chemistry

Additional Journal Information:

Journal Volume: 291; Journal Issue: 18; Journal ID: ISSN 0021-9258

Publisher:

American Society for Biochemistry and Molecular Biology

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; microscopy; parasite; peptidase; RNA interference (RNAi); Trypanosoma brucei