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.
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 vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.
Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.|Immunogenetic Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Somayeh
S
Sadeghi
Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
Negar
N
Seyed
Department of Virology, Pasteur Institute of Iran, Tehran, Iran.
Mohammad-Hossein
MH
Etemadzadeh
Immunogenetic Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.
Saeid
S
Abediankenari
Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
Sima
S
Rafati
Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran, Tehran, Iran.
Tahereh
T
Taheri
D000666:Amphotericin B / Q000494:pharmacology*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods*; D005260:Female; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018320:Leishmania major / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D016773:Leishmaniasis, Cutaneous / Q000469:parasitology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice
D000981:Antiprotozoal Agents; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000666:Amphotericin B; D008156:Luciferases
EGFP; Leishmania major; MTT; amphotericin B; luciferase; nitric oxide