Centre for Immunology and Infection, Department of Biology, University of York, York, YO10 5DD, UK.
Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK.
Parasit Vectors. 2020 Mar 14;13(1):132. doi: 10.1186/s13071-020-3999-1.
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.
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.
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.
NMT is an essential gene in all parasite life-cycle stages, confirming its role as a genetically-validated target for drug development.
证明特定基因对于巨噬细胞内利什曼原虫寄生虫的细胞内存活是必需的，这仍然是鉴定适合药物开发的靶标的挑战。在缺乏强大的诱导表达系统或在所有利什曼物种中都能发挥作用的功能 RNAi 机制的情况下，这一点尤为明显。目前，如果一个感兴趣的靶基因在细胞外寄生虫中只能在异位表达野生型拷贝的情况下从其基因组座点中缺失，那么可以假设该基因对于促进疾病的细胞内寄生虫的存活也是必需的。然而，为了稳健地验证感兴趣的基因作为化学干预的潜在靶标，必须在两个生命周期阶段中独立证明其功能的必需性。
在这里，我们使用质粒洗牌方法在体内提供了支持性遗传证据，证明 N-豆蔻酰转移酶（NMT）在寄生虫整个生命周期中对利什曼原虫的存活是必需的。在证实了载体传播的前鞭毛体中的 NMT 必需性后，使用一系列突变寄生虫在进行负选择压力之前感染小鼠，以检验 NMT 对于已建立感染中的寄生虫存活也是必需的假设。
只有在存在另一个拷贝的情况下，异位表达的 NMT 才是可有可无的。仅当受到负选择影响的另一个 NMT 拷贝表达时，接受负选择的动物中的寄生虫总负担才与对照组相当。
NMT 是所有寄生虫生命周期阶段的必需基因，证实了它作为药物开发的遗传验证靶标的作用。
