The role of Trypanosoma brucei MRPA in melarsoprol susceptibility
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:
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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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2019, Journal of Global Antimicrobial ResistanceCitation Excerpt :This localisation of TbMRPE possibly confers resistance via a sequestration mechanism. Moreover, an increased level of trypanothione and glutathione in T. brucei results in a slight increase of TbMRPA-mediated melarsen resistance through overexpression of ornithine decarboxylase or γ-glutamyl cysteine synthetase, whereas in melarsen-resistant cell lines trypanothione or glutathione levels are unchanged [117,118]. Therefore, in T. brucei the level of endogenous glutathione/trypanothione is sufficient to conjugate melarsen as well as other drugs.
Melarsoprol Resistance in African Trypanosomiasis
2018, Trends in ParasitologyCitation Excerpt :Resistance was found to be due to reduced drug uptake for both melarsen oxide [45] and pentamidine [46,47]. Arsenical efflux is another possible mechanism of resistance, and overexpression of the ABC transporter multidrug resistance protein A (MRPA) does indeed result in melarsoprol resistance [48]. This was not the case in a mouse model, however, and no MRPA overexpression was detected in melarsoprol-resistant isolates from HAT patients [48].
Drugs and Drug Resistance in African and American Trypanosomiasis
2018, Annual Reports in Medicinal ChemistryGenomic analysis of Isometamidium Chloride resistance in Trypanosoma congolense
2017, International Journal for Parasitology: Drugs and Drug ResistanceCitation Excerpt :Members of the DMT superfamily have been involved in antibiotics resistance in Staphylococcus aureus (Grinius and Goldberg, 1994) and Escherichia coli (Yerushalmi et al., 1995), and in chloroquine resistance in P. falciparum (Martin and Kirk, 2004). Similarly, an important role of ABC transporters in drug resistance has previously been proposed in T. congolense (Delespaux et al., 2005) and has been documented in many other organisms including T. brucei, T. cruzi, P. falciparum, Schistosoma mansoni, and Leishmania parasites (Ouellette et al., 2001; Perez-Victoria et al., 2002; Shahi et al., 2002; Alibu et al., 2006; Sanderson et al., 2009; Franco et al., 2015; Garg and Goyal, 2015; Pinto-Almeida et al., 2015; Veiga et al., 2016). Changes in other transport proteins also conferred resistance in Trypanosoma spp. (Barrett et al., 1995; Matovu et al., 2003; de Koning et al., 2004; Witola et al., 2004; Munday et al., 2015).
The promiscuous binding of pharmaceutical drugs and their transporter-mediated uptake into cells: What we (need to) know and how we can do so
2013, Drug Discovery TodayCitation Excerpt :In addition, both pentamidine and melarsoprol are also transported via the adenosine transporter AT1 [194–200], pentamidine is transported using NT11.1 and NT12.1 [201] while the source of energy for concentrative pentamidine uptake is provided by three H+-ATPases HA1–3 [190] (a mitochondrial pentamidine uptake carrier is not yet known). Both melarsoprol [202] and pentamidine are also substrates for the multidrug ABC efflux transporter MRPA [203]. Thus we find multiple transporters capable of (with at least some being functionally necessary for) the transport of (and or resistance to) either or both of the antitrypanosomal drugs melarsoprol and pentamidine, a fact of considerable and demonstrable significance in the development of drug resistance in the target organisms.
- 1
These authors contributed equally to this work.
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Present address: Department of Pharmacology, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.