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JAC Advance Access published online on June 12, 2009
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkp206
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© The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Transgenic Leishmania donovani clinical isolates expressing green fluorescent protein constitutively for rapid and reliable ex vivo drug screening

Nasib Singh1, Reema Gupta1, Anil K. Jaiswal1, Shyam Sundar2 and Anuradha Dube1,*

1 Division of Parasitology, Central Drug Research Institute, Lucknow, India 2 Department of Medicine, Institute of Medical Sciences, BHU, Varanasi, India

Received 19 March 2009; returned 8 May 2009; revised 18 May 2009; accepted 19 May 2009

* Corresponding author. Tel: +91-522-2612411/2612418, ext. 4398; Fax: +91-522-2623938/2623405; E-mail: anuradha_dube{at}hotmail.com

Objectives: Several Leishmania strains with episomal expression of green fluorescent protein (GFP) require constant drug pressure for its continuous expression and hence limit its use in ex vivo or in vivo systems. The aim of this study was to alleviate this problem by stably integrating the GFP gene into the parasite genome, so as to use these transfectants for ex vivo and in vivo drug screening.

Methods: The GFP gene was integrated downstream of the 18S ribosomal promoter region of Leishmania donovani. After initial selection, GFP-expressing parasites—both sodium stibogluconate (SAG)-susceptible (2001) and -resistant (2039) isolates—were grown without adding G418. The infectivity of these transfectants to macrophages (J774.1) as well as to hamsters was checked. The ex vivo screening assay was standardized using standard antileishmanial drugs.

Results: A constitutive and enhanced expression of GFP in promastigote and amastigote stages was achieved for ~12 months without any need for drug pressure. These transfectants were highly infective to macrophage cell lines as well as to hamsters, as observed by fluorescence microscopy and flow cytometry (FACS). GFP-tagged promastigotes as well as intracellular amastigotes were found to be highly susceptible to miltefosine, amphotericin B and pentamidine, in a concentration-dependent manner. SAG was inactive against the GFP-promastigotes, as well as SAG-resistant intracellular amastigotes, correlating well with earlier reports.

Conclusions: The GFP-transfectants were found to be suitable for FACS-based ex vivo screening assays. They were also infective to hamsters up to day 60 post-infection.

Key Words: stable integration , GFP , Leishmania clinical isolates , hamsters , ex vivo antileishmanial screening


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