Daniel Paape1,2, Catriona T Prendergast3,4, Helen P Price3,5, Johannes S P Doehl3, Deborah F Smith3. 1. Centre for Immunology and Infection, Department of Biology, University of York, York, YO10 5DD, UK. daniel.paape@glasgow.ac.uk. 2. Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK. daniel.paape@glasgow.ac.uk. 3. Centre for Immunology and Infection, Department of Biology, University of York, York, YO10 5DD, UK. 4. Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK. 5. Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Newcastle-under-Lyme, ST5 5BG, UK.
Abstract
BACKGROUND: 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. 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 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. 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 affected by the negative selection, was expressed. CONCLUSIONS: NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.
BACKGROUND: 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. 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 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. 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 affected by the negative selection, was expressed. CONCLUSIONS:NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.
Authors: Andrew S Bell; Zhiyong Yu; Jennie A Hutton; Megan H Wright; James A Brannigan; Daniel Paape; Shirley M Roberts; Charlotte L Sutherell; Markus Ritzefeld; Anthony J Wilkinson; Deborah F Smith; Robin J Leatherbarrow; Edward W Tate Journal: J Med Chem Date: 2020-07-14 Impact factor: 7.446