Abstract
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.
Highlights
The leishmaniases are a spectrum of diseases caused by infection with protozoan pathogens of the Leishmania genus, with an estimated 1 million new cases per annum. [1] Leishmania parasites are transmitted to a mammalian host via the bite of an infected sand fly
The protozoan parasite Leishmania causes a spectrum of diseases collectively known as leishmaniasis
Leishmaniasis is classified as a neglected tropical disease, and is endemic in 97 countries
Summary
The leishmaniases are a spectrum of diseases caused by infection with protozoan pathogens of the Leishmania genus, with an estimated 1 million new cases per annum. [1] Leishmania parasites are transmitted to a mammalian host via the bite of an infected sand fly. The Drugs for Neglected Diseases initiative (DNDi) was set up to identify potential lead compounds This has yielded success with the identification of three new chemical series that display considerable anti-leishmanial potential. Efficient compound screening requires robust, sensitive and reproducible assays that are suitable for high throughput application. [6,7,8] The main argument for phenotypic assays is that they directly measure compound activity against the target cell This tends to be determined using either a colourmetric or fluorescent regent that measures metabolism (e.g. MTT [9] or AlamarBlue [10]), or using a fluorescent reporter molecule such as GFP [11,12,13] or mCherry. Parasite-specific fluorescent reporter molecules show the presence and localisation of parasites; the assays used tend to look for the presence of the fluorescent parasite (either by microscopy or flow cytometry), not parasite viability
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