Efflux Systems and Increased Trypanothione Levels in Arsenite-Resistant Leishmania Danielle Le ´gare ´,* Barbara Papadopoulou,* Gae ´tan Roy,* Rita Mukhopadhyay,† Anass Haimeur,* Saibal Dey,† Katherine Grondin,* Christian Brochu,* Barry P. Rosen,† and Marc Ouellette* ,1 *Centre de Recherche en Infectiologie du Centre de Recherche du CHUL et De ´partement de Microbiologie, Faculte ´ de Me ´decine, Universite ´ Laval, Que ´bec, Canada; and †Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201, U.S.A. Le ´gare ´, D., Papadopoulou, B., Roy, G., Mukhopadhyay, R., Haimeur, A., Dey, S., Grondin, K., Brochu, C., Rosen, B. P., and Ouellette, M. 1997. Efflux systems and increased trypanothione levels in arsenite-resistant Leishmania. Experimental Parasitology 87, 275–282. The mechanism of resis- tance to the metal arsenite has been studied and compared in L. mexicana, L. tropica, and L. tarentolae selected in a step by step manner for arsenite resistance. Amplification of the ABC transporter gene pgpA was found to be a frequent resistance mechanism in all species. Transfection of pgpA genes into different species indicated that both the origin of the pgpA gene and the recipient strain into which the gene is transfected seem important for resistance. An increase in the levels of trypanothione was also correlated with metal resistance in different Leishmania species. The mecha- nism used to increase the levels of trypanothione seems to differ, however, between the different species. This study points to a key role of transporters and thiol levels in metal resistance in Leishmania. © 1997 Academic Press Index Descriptors and Abbreviations: ABC transporters; Leishmania; trypanothione; efflux sys- tems; heavy metals; GSH, glutathione; TSH, trypanothione; -GCS, -glutamylcysteine synthetase; ABC, ATP binding cassette. INTRODUCTION Arsenic and antimony containing drugs are still the first line of choice in the treatment of some parasitic diseases. For example, pentava- lent antimony is used against most infections caused by the protozoan parasite Leishmania. Leishmania cells resistant to antimonials are en- countered more and more frequently (Jackson et al. 1990; Grogl et al. 1992; Faraut-Gambarelli et al. 1997) and an understanding of the resis- tance mechanism(s) is warranted. The rate of appearance of resistance in the field has been slow to arise, which suggests that resistance is the consequence of more than one mutation. We have championed the idea that the study of drug resistance mechanisms induced under labora- tory conditions may be helpful in deciphering complex resistance mechanisms present in field isolates (Borst and Ouellette, 1995). In our quest to understand metal resistance in Leish- mania, we have studied resistance to the related metal arsenite. Several clones of Leishmania tarentolae were selected in a step by step manner for re- sistance to arsenite but also to trivalent and pen- tavalent antimony (Dey et al. 1994). The first metal resistance gene characterized coded for the ABC transporter PgpA (Ouellette et al. 1990). The pgpA gene was found to be ampli- fied in strains of L. mexicana (Detke et al. 1989) and L. tarentolae cells (Ouellette et al. 1991) selected for arsenite resistance. Transfection ex- periments indicated that PgpA was indeed in- volved in metal resistance, although resistance levels varied depending on the species into which the gene was transfected (Callahan and Beverley, 1991; Papadopoulou et al. 1994a). Recently we showed that PgpA possibly recog- nizes the metal conjugated to trypanothione 1 To whom correspondence should be addressed at Ser- vice d’Infectiologie du CHUL, 2705 boul. Laurier, Ste-Foy, Que ´bec, Canada G1V 4G2. Fax: 418-654 2715. E-mail: Marc.Ouellette@crchul.ulaval.ca. EXPERIMENTAL PARASITOLOGY 87, 275–282 (1997) ARTICLE NO. PR974222 275 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.