Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (
egfp) gene or
β-galactosidase gene (
lacZ) were constructed for drug screening and histopathological analysis. The
egfp or
lacZ in a leishmanial transfection vector, p6.5, was introduced into
L. amazonensis promastigotes, and
egfp or
lacZ-carrying recombinant
L. amazonensis, La/
egfp and La/
lacZ, respectively, were obtained. Expression of
egfp or
lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-
β-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and
β-galactosidase activity measured by a colorimetric reaction with chlorophenol red
β-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC
50) of a leishmanicidal drug, amphotericin B, in
L. amazonensis promastigotes measured using La/
egfp and La/
lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/
egfp and La/
lacZ amastigotes in macrophages, respectively, suggesting La/
egfp and La/
lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of
L. amazonensis. La/
lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/
lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages
in vivo. Thus, the marker-transfected
Leishmania parasites constructed in this study will be useful for analyses of
Leishmania parasites, especially at the intracellular stage.
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