Development of NanoLuc-PEST expressing Leishmania mexicana as a new drug discovery tool for axenic- and intramacrophage-based assays

The protozoan parasite Leishmania causes leishmaniasis; a spectrum of diseases of which there are an estimated 1 million new cases each year. Current treatments are toxic, expensive, difficult to administer, and resistance to them is emerging. New therapeutics are urgently needed, however, screening the infective amastigote form of the parasite is challenging. Only certain species can be differentiated into axenic amastigotes, and compound activity against these does not always correlate with efficacy against the parasite in its intracellular niche. Methods used to assess compound efficacy on intracellular amastigotes often rely on microscopy-based assays. These are laborious, require specialist equipment and can only determine parasite burden, not parasite viability. We have addressed this clear need in the anti-leishmanial drug discovery process by producing a transgenic L. mexicana cell line that expresses the luciferase NanoLuc-PEST. We tested the sensitivity and versatility of this transgenic strain, in comparison with strains expressing NanoLuc and the red-shifted firefly luciferase. We then compared the NanoLuc-PEST luciferase to the current methods in both axenic and intramacrophage amastigotes following treatment with a supralethal dose of Amphotericin B. NanoLuc-PEST was a more dynamic indicator of cell viability due to its high turnover rate and high signal:background ratio. This, coupled with its sensitivity in the intramacrophage assay, led us to validate the NanoLuc-PEST expressing cell line using the MMV Pathogen Box in a two-step process: i) identify hits against axenic amastigotes, ii) screen these hits using our bioluminescence-based intramacrophage assay. The data obtained from this highlights the potential of compounds active against M. tuberculosis to be re-purposed for use against Leishmania. Our transgenic L. mexicana cell line is therefore a highly sensitive and dynamic system suitable for Leishmania drug discovery in axenic and intramacrophage amastigote models.

原生动物寄生虫利什曼原虫（Leishmania）可引发利什曼病（leishmaniasis），这是一类疾病谱，据估算每年新增约100万例病例。当前治疗方案存在毒性强、成本高昂、给药难度大的问题，且耐药性正不断出现。目前亟需开发新型治疗药物，但筛选该寄生虫的感染性无鞭毛体（amastigote）形式极具挑战。仅特定物种可被诱导分化为无菌培养无鞭毛体（axenic amastigote），且针对此类无鞭毛体的化合物活性，并不总能与其在细胞内寄生微环境中对抗寄生虫的效力相匹配。当前用于评估细胞内无鞭毛体化合物效力的检测方法多以显微镜技术为基础，此类操作繁琐、需专用实验设备，且仅能检测寄生虫载量，无法评估寄生虫的活力。为解决抗利什曼病药物研发中的这一明确需求，我们构建了表达荧光素酶NanoLuc-PEST的转基因墨西哥利什曼原虫（L. mexicana）细胞系。我们将该转基因菌株与分别表达NanoLuc和红移型萤火虫荧光素酶（red-shifted firefly luciferase）的菌株进行对比，评估了其灵敏度与通用性。随后，我们在无菌培养无鞭毛体与巨噬细胞内寄生无鞭毛体两种模型中，以超致死剂量的两性霉素B（Amphotericin B）处理样本，将NanoLuc-PEST荧光素酶检测法与现有方法进行了对比。NanoLuc-PEST凭借其高周转速率与高信号背景比，可更动态地反映细胞活力。结合其在巨噬细胞内检测中的优异灵敏度，我们采用MMV病原体库（MMV Pathogen Box），通过两步流程对该表达NanoLuc-PEST的细胞系进行了验证：其一，筛选出对无菌培养无鞭毛体具有活性的命中化合物；其二，利用我们的生物发光巨噬细胞内检测方法，对上述活性化合物进行复筛。本次研究获得的数据表明，对结核分枝杆菌（M. tuberculosis）具有活性的化合物，有望被重新开发用于抗利什曼病治疗。综上，我们构建的转基因墨西哥利什曼原虫细胞系是一种高灵敏度、高动态性的检测系统，可适用于无菌培养无鞭毛体与巨噬细胞内寄生无鞭毛体两种模型中的利什曼病药物研发工作。