Paape, D, Prendergast, CT, Price, HP ORCID: https://orcid.org/0000-0003-1537-4390, Doehl, JSP and Smith, DF (2020) Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model. Parasites & Vectors, 13 (1).

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Abstract

Background: Proving that specifc genes are essential for the intracellular viability of Leishmania parasites within
macrophages remains a challenge for the identifcation of suitable targets for drug development. This is especially
evident in the absence of a robust inducible expression system or functioning RNAi machinery that works in all Leishmania species. Currently, if a target gene of interest in extracellular parasites can only be deleted from its genomic
locus in the presence of ectopic expression from a wild type copy, it is assumed that this gene will also be essential for
viability in disease-promoting intracellular parasites. However, functional essentiality must be proven independently
in both life-cycle stages for robust validation of the gene of interest as a putative target for chemical intervention.
Methods: Here, we have used plasmid shufe methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confrmation
of NMT essentiality in vector-transmitted promastigotes, a range of mutant parasites were used to infect mice prior
to negative selection pressure to test the hypothesis that NMT is also essential for parasite viability in an established
infection.
Results: Ectopically-expressed NMT was only dispensable under negative selection in the presence of another copy.
Total parasite burdens in animals subjected to negative selection were comparable to control groups only if an additional NMT copy, not afected by the negative selection, was expressed.
Conclusions: NMT is an essential gene in all parasite life-cycle stages, confrming its role as a genetically-validated
target for drug development.
Keywords: Leishmania, Plasmid shufe, Mouse infection, Therapeutic target validation

Item Type: Article
Additional Information: Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Uncontrolled Keywords: Genetic, Leishmania genes, amastigote, vivo, N-myristoyltransferase .
Subjects: Q Science > Q Science (General)
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Divisions: Faculty of Natural Sciences > School of Life Sciences
Depositing User: Symplectic
Date Deposited: 18 Mar 2020 15:14
Last Modified: 18 Mar 2020 15:14
URI: http://eprints.keele.ac.uk/id/eprint/7808

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