The role of Trypanosoma brucei MRPA in melarsoprol susceptibility

https://doi.org/10.1016/j.molbiopara.2005.10.016Get rights and content

Abstract

We previously showed that over-expression of Trypanosoma brucei MRPA, a member of the multidrug resistance protein family in T. brucei, reproducibly resulted in resistance to the anti-trypanosomal drug melarsoprol in vitro. MRPA is predicted to mediate efflux of melarsoprol as a conjugate with trypanothione, a glutathione–spermidine conjugate which is the major small thiol in trypanosomes. Here, we show that depletion of MRPA by RNA interference resulted in moderate hypersensitivity to both melarsoprol and melarsen oxide. Over-expression of MRPA alone is not sufficient to cause melarsoprol resistance in vivo, although it is sufficient in vitro. This discrepancy is not an effect of drug metabolism since over-expression of MRPA alone conferred resistance to melarsoprol and its principle metabolite, melarsen oxide, in vitro. Over-expression of MRPA was not detected in four melarsoprol-resistant trypanosome isolates from sleeping sickness patients.

Introduction

Only two drugs are currently available for the treatment of late-stage African sleeping sickness, which is invariably fatal if untreated. One, eflornithine (DFMO), is active only against the West African form of the disease, caused by Trypanosoma brucei gambiense, while the other, a melaminophenyl arsenical called melarsoprol, is active against both T. b. gambiense and the East African variant, Trypanosoma brucei rhodesiense [1]. Resistance to melarsoprol is increasing in the field and there is currently no alternative treatment for late-stage T. b. rhodesiense infection [2].

Melarsoprol enters trypanosomes partly by passive diffusion across the parasite membrane, and partly through active uptake mediated by the adenosine transporter, TbATl [3]. Previous work has shown that melarsoprol resistance can be caused by inactivating mutations or deletion of the TbATl gene [4], [5], [6], [7]. Two observations suggest, however, that TbATl mutations may not be the sole cause of melarsoprol resistance. First, only about 50% of clinically resistant field isolates appear to have the inactivating mutations [8], [9]. Second, deletion of the TbATl gene results in only two- to three-fold melarsoprol resistance in vitro [6]; although this may be sufficient for clinical failure, isolates from the field often show considerably higher levels of melarsoprol insensitivity [2]. We have therefore looked for alternative mechanisms of melarsoprol resistance.

Leishmania species can become resistant to arsenite or antimonial drugs through gene amplification and consequent increased expression of a multidrug-resistance protein (MRP) called PGPA, which transports metal–thiol conjugates [10]. The major small thiol in T. brucei is trypanothione, a conjugate of glutathione with spermidine, and to obtain drug resistance in Leishmania increased production of trypanothione is required [11], [12], [13], [14], [15].

We previously showed that over-expression of the T. brucei MRPA homologue in T. brucei can cause a 10-fold increase in resistance to melarsoprol in vitro. In contrast to the results from Leishmania, over-expression of two enzymes of trypanothione biosynthesis, ornithine decarboxylase (ODC) and gamma glutathionyl spermidine synthase (GCS), enhanced the resistance only slightly [16]. In this paper we addressed two questions: does MRPA over-expression contribute to melarsoprol treatment failures in sleeping sickness patients, and what are the effects of down-regulation of MRPA expression?

Section snippets

Trypanosome culture and transfection

The cell lines used for in vivo experiments were described previously [16]. For all other experiments, bloodstream trypanosomes of the Lister 427 strain, constitutively expressing T7 polymerase and the tet repressor, were used [17]. For MRPA-myc expression, the cells were transfected with Not I-linearized pHD 1057 as previously described [16]. This plasmid contains, in addition to a pGEM backbone, the following elements:

  • 1.

    A segment from a non-transcribed intergenic spacer of a ribosomal RNA gene

Effect of MRPA over-expression on melarsoprol sensitivity in vivo

We first tested whether over-expression of MRPA results in drug resistance in vivo. To do this, we used T. brucei (strain 927) bloodstream forms which were described previously [16]. The parasites tested over-express various transgenes: MRPA and two genes involved in trypanothione biosynthesis, ODC and GCS. In addition, we used cells over-expressing MRPE, an MRP-like putative transporter whose over-expression does not cause melarsoprol resistance in vitro [16]. These cells were used to infect

Discussion

Results from our previous experiments [16] and those described in this paper indicate that over-expression of TbMRPA alone can cause arsenical resistance in trypanosomes in vitro. We were therefore surprised to find that such cells were not resistant to melarsoprol in vivo. We initially thought that the difference between the in vitro and the in vivo experiments might be caused by in vivo metabolism of melarsoprol to melarsen oxide. Since, however, the MRPA-expressing cells were resistant to

Acknowledgements

We thank W. Wickstead for the p2T7TA-177 vector and S. Hänni, University of Bern, and A. Scheider, University of Fribourg, for the pSLcompl stem-loop vector. VPA was supported by the UNDP/World Bank/World Health Organisation Program for Research and Training in Tropical Diseases (TDR), and GM and TS acknowledge support from the Schweizer Nationalfonds. We acknowledge the technical assistance of Guy Riccio (STI) with the in vivo mouse work. CR expressed MRPA (assisted by SS), generated the

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    1

    These authors contributed equally to this work.

    2

    Present address: Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.

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