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.

中文翻译：

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使用 N-肉豆蔻酰转移酶作为模型对体内无鞭毛体生存必需的利什曼原虫基因进行遗传验证


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生存能力至关重要，这对于确定药物开发的合适靶点仍然是一个挑战。在缺乏适用于所有利什曼原虫物种的强大的诱导表达系统或功能性 RNAi 机制的情况下，这一点尤其明显。目前，如果细胞外寄生虫中感兴趣的靶基因只能在存在野生型拷贝的异位表达的情况下从其基因组基因座中删除，则假设该基因对于促进疾病的细胞内寄生虫的生存能力也至关重要。然而，必须在两个生命周期阶段独立证明功能重要性，以强有力地验证感兴趣的基因作为化学干预的假定目标。在这里，我们在体内使用质粒改组方法来提供支持性遗传证据，证明 N-肉豆蔻酰转移酶 (NMT) 对于利什曼原虫在整个寄生虫生命周期中的生存能力至关重要。在确认 NMT 在载体传播的前鞭毛体中的重要性后，在负选择压力之前使用一系列突变寄生虫感染小鼠，以测试 NMT 对于已建立的感染中的寄生虫活力也至关重要的假设。异位表达的 NMT 仅在存在另一个拷贝的负选择下才是可有可无的。仅当表达不受负选择影响的额外 NMT 拷贝时，经受负选择的动物中的总寄生虫负荷才与对照组相当。 NMT 是寄生虫生命周期所有阶段的重要基因，证实了其作为药物开发的基因验证靶标的作用。