In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

The Korean Journal of Parasitology
Somayeh SadeghiTahereh Taheri

Abstract

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitr...Continue Reading

References

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Citations

Dec 22, 2015·Applied Microbiology and Biotechnology·Samira SeifTahereh Taheri
Jul 15, 2017·PLoS Neglected Tropical Diseases·Alireza BadirzadehSima Rafati
Dec 19, 2017·PLoS Neglected Tropical Diseases·Zahra AbdossamadiSima Rafati
Nov 26, 2018·Parasite Immunology·Maryam Heidari-KharajiMehrak Zare
Apr 28, 2020·Pathogens and Global Health·Sofia CortesLenea Campino

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Methods Mentioned

BETA
fluorescence-activated cell sorting
hi-FCS
PMA
flow cytometry
protein folding

Software Mentioned

FlowJo
GraphPad Prism

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