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Antimicrobial Agents and Chemotherapy, April 2001, p. 1121-1125, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1121-1125.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

In Vivo Monitoring of Intracellular ATP Levels in Leishmania donovani Promastigotes as a Rapid Method To Screen Drugs Targeting Bioenergetic Metabolism

Juan Román Luque-Ortega,1 Octavio Miguel Rivero-Lezcano,1 Simon L. Croft,2 and Luis Rivas1,*

Centro de Investigaciones Biológicas, CSIC, Velázquez 144, E-28006, Madrid, Spain,1 and Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London WC1E 7HT, United Kingdom2

Received 31 July 2000/Returned for modification 2 October 2000/Accepted 18 January 2001

A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralis luciferase and incubated with free-membrane permeable D-luciferin analogue D-luciferin-[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (Delta Psi m) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 µM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.

* Corresponding author. Mailing address: Centro de Investigaciones Biológicas (C.S.I.C.), Velázquez 144, E-28006, Madrid, Spain. Phone: (34)915611800-4234. Fax: (34)91-5627518. E-mail: luis_rivas{at}cib.csic.es.

Antimicrobial Agents and Chemotherapy, April 2001, p. 1121-1125, Vol. 45, No. 4
0066-4804/01/$04.00+0   DOI: 10.1128/AAC.45.4.1121-1125.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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