High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric beta-lactamase assay.
A simple colorimetric beta-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The beta-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with beta-Lactamase gene. These beta-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTA beta-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.
Mandal
Swati
S
School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India.
Maharjan
Mahendra
M
Ganguly
Sudipto
S
Chatterjee
Mitali
M
Singh
Sarman
S
Buckner
Frederick S
FS
Madhubala
Rentala
R
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology / Q000627:therapeutic use; D001681:Biological Assay / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D007893:Leishmania donovani / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016773:Leishmaniasis, Cutaneous / Q000188:drug therapy; D007898:Leishmaniasis, Visceral / Q000188:drug therapy; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D000981:Antiprotozoal Agents; D001618:beta-Lactamases