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

Daniel Paape,Johannes S P Doehl,Helen P Price,Deborah F Smith,Catriona T Prendergast

doi:10.1186/s13071-020-3999-1

Abstract

BackgroundProving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification 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.MethodsHere, we have used plasmid shuffle methods in vivo to provide supportive genetic evidence that N-myristoyltransferase (NMT) is essential for Leishmania viability throughout the parasite life-cycle. Following confirmation 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.ResultsEctopically-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 affected by the negative selection, was expressed.ConclusionsNMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.

Highlights

Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification of suitable targets for drug development
The second plasmid was engineered so that the tdTomato was under the same regulatory control as green fluorescent protein (GFP) in the NMT-thymidine kinase (TK) plasmid
Plasmid shuffle [31] provides an alternative approach for the positive discrimination of essential genes as it involves the deletion of the gene of interest from its genomic locus in the presence of an ectopic copy, but testing of whether this ectopic copy is dispensable upon negative selection [28,29,30]

Summary

Introduction

Proving that specific genes are essential for the intracellular viability of Leishmania parasites within macrophages remains a challenge for the identification 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. 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. The kinetoplast parasites, Leishmania spp., alternate between two distinct life-cycle stages: the flagellated and motile extracellular promastigotes, and the immotile intracellular amastigotes bearing their rudimentary flagellum [1, 2]. These challenges are obviated in target-based screening, where both the target identity and its mode of action can be studied in detail, leading to compound optimisation guided by structural constraints and definition of a structure activity relationship [12, 13]

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Conclusion