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Acquired resistance to berenil in a cloned isolate of Trypanosoma evansi is associated with upregulation of a novel gene, TeDR40

  • W. H. WITOLA (a1), A. TSUDA (a1), N. INOUE (a2), K. OHASHI (a1) and M. ONUMA (a1)...


Drug resistance is now a severe and increasing problem in trypanosomes, but molecular details of mechanisms of resistance are only beginning to unveil. There is urgent need to clearly elucidate the different mechanisms of drug resistance in trypanosomes in order to circumvent existing resistance problems and avoid emergence of resistance to the next generation drugs. In this study, we cloned and characterized a novel gene, TeDR40, whose expression is associated with resistance to berenil in Trypanosoma evansi. Expression analysis showed that the gene was at least 1000-fold upregulated in resistant parasites and the encoded protein appeared to have a ubiquitous cellular localization. To investigate the association of TeDR40 with berenil-resistance, we genetically modified wild-type berenil-sensitive T. evansi for inducible over-expression of the TeDR40 gene. Induction of over-expression of TeDR40 in T. evansi led to decreased (P<0·01) sensitivity to berenil. Our findings indicate a possible correlation between over-expression of a novel gene, TeDR40, and reduced sensitivity to berenil in an in vitro-cultured clonal line of T. evansi.


Corresponding author

Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. Tel: +81 11 7065215. Fax: +81 11 7065217. E-mail:


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Anene, B. M., Onah, D. N. and Nawa, Y. ( 2001). Drug resistance in pathogenic African trypanosomes: what hopes for the future? Veterinary Parasitology 96, 83100.
Barret, M. P., Zhang, Z. Q., Denise, H., Giroud, C. and Baltz, T. ( 1995). A diamidine-resistant Trypanosoma equiperdum clone contains a P2 purine transporter with reduced substrate affinity. Molecular and Biochemical Parasitology 73, 223229.
Benghezal, M., Benachour, A., Rusconi, S., Aebi, M. and Conzelmann, A. ( 1996). Yeast Gpi8p is essential for GPI anchor attachment onto proteins. European Molecular Biology Organization Journal 15, 65756583.
Beverley, S. M., Coderre, J. A., Santi, D. V. and Schimke, R. T. ( 1984). Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell 38, 431439.
Carruthers, V. B. and Cross, G. A. M. ( 1992). High-efficiency clonal growth of bloodstream and insect-form Trypanosoma brucei on agarose plates. Proceedings of the National Academy of Sciences, USA 89, 88188821.
Carter, N. S., Berger, B. J. and Fairlamb, A. H. ( 1995). Uptake of diamidine drugs by the P2 nucleoside transporter in melarsen-sensitive and -resistant Trypanosoma brucei brucei. Journal of Biological Chemistry 270, 2815328157.
Cross, G. A. M. ( 1990). Glycolipid anchoring of plasma membrane proteins. Annual Reviews of Cell Biology 6, 139.
De Greef, C. and Hamers, R. ( 1994). The serum resistance-associated (SRA) gene of Trypanosoma brucei rhodesiense encodes a variant surface glycoprotein-like protein. Molecular and Biochemical Parasitology 68, 277284.
De Greef, C., Imberechts, H., Matthyssens, G., Van Meirvenne, N. and Hamers, R. ( 1989). A gene expressed only in serum-resistant variants of Trypanosoma brucei rhodesiense. Molecular and Biochemical Parasitology 36, 169176.
De Koning, H. P., Anderson, L. F., Stewart, M., Burchmore, R. J. S., Wallace, L. J. M. and Barret, M. P. ( 2004). The trypanocide diminazene aceturate is accumulated predominantly through the TbAT1 purine transporter: additional insights on diamidine resistance in African trypanosomes. Antimicrobial Agents and Chemotherapy 48, 15151519.
De Koning, H. P. and Jarvis, S. M. ( 1999). Adenosine transporters in bloodstream forms of Trypanosoma brucei brucei: substrate recognition motifs and affinity for trypanocidal drugs. Molecular Pharmacology 56, 11621170.
De Koning, H. P. ( 2001). Uptake of pentamidine in Trypanosoma brucei is mediated by three distinct transporters: implications for cross-resistance with arsenicals. Molecular Pharmacology 59, 586592.
Diatchenko, L., Chenchik, A. and Siebert, P. ( 1998). Suppression subtractive hybridization: A method for generating subtracted cDNA libraries starting from poly (A)+ or total RNA. In RT-PCR Methods for Gene Cloning and Analysis ( ed. Siebert, P. and Larrick, J.), pp. 213239. BioTechniques Books, MA, USA.
Ehrlich, P. ( 1907). Chemotherapeutische Trypanosomen-Studien. Berliner klinische Wochenschrift 11, 310314.
Englund, P. T. ( 1993). The structure and biosynthesis of glycosylphosphatidylinositol protein anchors. Annual Reviews of Biochemistry 62, 121138.
Ferguson, M. A. J. ( 1999). The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research. Journal of Cell Science 112, 27992809.
Fraering, P., Imhof, I., Meyer, U., Strub, J. M., van Dorsselaer, A., Vionnet, C. and Conzelmann, A. ( 2001). The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p and Gpi16p. Molecular Biology of the Cell 12, 32953306.
Geerts, S., Holmes, P. H., Diall, O. and Eisler, M. C. ( 2001). African bovine trypanosomiasis: the problem of drug resistance. Trends in Parasitology 17, 2528.
Graham, S. V. ( 1995). Mechanisms of stage-regulated gene expression in kinetoplastida. Parasitology Today 11, 217223.
Gurskaya, N. G., Diatchenko, L., Chenchik, A., Siebert, P. D., Khaspekov, G. L., Lukyanov, K. A., Vagner, L. L., Ermolaeva, O. D., Lukyanov, S. A. and Sverdlov, E. D. ( 1996). Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: Cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate. Analytical Biochemistry 240, 9097.
Hirumi, H. and Hirumi, K. ( 1989). Continuous cultivation of Trypanosoma brucei bloodstream forms in a medium containing a low concentration of serum protein without feeder cell layers. Journal of Parasitology 75, 985989.
Laemmli, U. K. ( 1970). Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, 680685.
Maser, P. and Kaminsky, R. ( 1998). Identification of three ABC transporter genes in Trypanosoma brucei spp. Parasitology Research 84, 106111.
Matovu, E., Geiser, F., Schneider, V., Maser, P., Enyaru, J. C. K., Kaminsky, R., Gallati, S. and Seebeck, T. ( 2001). Genetic variants of the TbAT1 adenosine transporter from African trypanosomes in relapse infections following melarsoprol therapy. Molecular and Biochemical Parasitology 117, 7381.
Matovu, E., Stewart, M. L., Geiser, F., Brun, R., Maser, P., Wallace, L. J. M., Burchmore, R. J., Enyaru, J. C. K., Barret, M. P., Kaminsky, R., Seebeck, T. and De Koning, H. P. ( 2003). Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei. Eukaryotic Cell 2, 10031008.
Meyer, U., Benghezal, M., Imhof, I. and Conzelmann, A. ( 2000). Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. Biochemistry 39, 34613471.
Ohishi, K., Inoue, N. and Kinoshita, T. ( 2001). PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8. European Molecular Biology Organization Journal 20, 40884098.
Pays, E., Coquelet, H., Tebabi, P., Pays, A., Jefferies, D., Steinert, M., Koening, E., Williams, R. O. and Roditi, I. ( 1990). Trypanosoma brucei: constitutive activity of the VSG and procyclin gene promoters. European Molecular Biology Organization Journal 9, 31453151.
Roditi, I. ( 1996). The VSG-procyclin switch. Parasitology Today 12, 4749.
Ross, C. A. and Barns, A. M. ( 1996). Alteration to one of the adenosine transporters is associated with resistance to Cymelarsen in Trypanosoma evansi. Parasitology Research 82, 183188.
Sambrook, J., Fritsch, E. F. and Maniatis, T. ( 1989). Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Shahi, S. K., Krauth-Siegel, R. L. and Clayton, C. E. ( 2002). Overexpression of putative thiol conjugate transporter TbMRPA causes melarsoprol resistance in Trypanosoma brucei. Molecular Microbiology 43, 11291138.
Sharma, D. K., Hilley, J. D., Bangs, J. D., Coombs, G. H., Mottram, J. C. and Menon, A. K. ( 2000). Soluble GP18 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins. The Biochemical Journal 351, 717722.
Vanhamme, L. and Pays, E. ( 1995). Control of gene expression in trypanosomes. Microbiological Reviews 59, 223240.
Vidugiriene, J., Vainauskas, S., Johnson, A. E. and Menon, A. K. ( 2001). Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system. European Journal of Biochemistry 268, 22902300.
White, T. C., Fase-Fowler, F., van Luenen, H., Calafat, J. and Borst, P. ( 1988). The H circles of Leishmania tarentolae are a unique amplifiable system of oligomeric DNAs associated with drug resistance. Journal of Biological Chemistry 263, 1697716983.
Wilson, K., Berens, R. L., Sifri, C. D. and Ullman, B. ( 1994). Amplification of the inosinate dehydrogenase gene in Trypanosoma brucei gambiense due to an increase in chromosome copy number. Journal of Biological Chemistry 269, 2897928987.
Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. M. ( 1999). A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Molecular and Biochemical Parasitology 99, 89101.
Witola, W. H., Inoue, N., Ohashi, K. and Onuma, M. ( 2004). RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate. Experimental Parasitology 107, 4757.
Zhang, Z. Q., Giroud, C. and Baltz, T. ( 1993). Trypanosoma evansi: in vivo and in vitro determination of trypanocide resistance profiles. Experimental Parasitology 77, 387394.


Acquired resistance to berenil in a cloned isolate of Trypanosoma evansi is associated with upregulation of a novel gene, TeDR40

  • W. H. WITOLA (a1), A. TSUDA (a1), N. INOUE (a2), K. OHASHI (a1) and M. ONUMA (a1)...


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