Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model

Paape, D; Prendergast, CT; Price, H; Doehl, JSP; Smith, DF

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.

Citation

Paape, D., Prendergast, C., Price, H., Doehl, J., & Smith, D. (2020). Genetic validation of Leishmania genes essential for amastigote survival in vivo using N-myristoyltransferase as a model. Parasites and Vectors, 13, Article 132. https://doi.org/10.1186/s13071-020-3999-1

• Journal Article Type: Article
• Acceptance Date: Feb 26, 2020
• Publication Date: Mar 14, 2020
• Journal: Parasites & Vectors
• Print ISSN: 1756-3305
• Publisher: BioMed Central
• Peer Reviewed: Peer Reviewed
• Volume: 13
• Article Number: 132
• DOI: https://doi.org/10.1186/s13071-020-3999-1
• Keywords: Genetic, Leishmania genes, amastigote, vivo, N-myristoyltransferase .
• Publisher URL: https://doi.org/10.1186/s13071-020-3999-1