Trypanosoma brucei RNA interference in the mammalian host

Molecular & Biochemical Parasitology 140 (2005) 127–131

Short communication

Trypanosoma brucei RNA interference in the mammalian host Laurence Lecordier1 , David Walgraffe1 , Sara Devaux, Philippe Poelvoorde, Etienne Pays, Luc Vanhamme∗ Laboratory of Molecular Parasitology, Institute for Molecular Biology and Medicine (IBMM), Free University of Brussels, 12 rue des Professeurs Jeener et Brachet, 6041 Gosselies, Belgium Received 1 October 2004; accepted 17 December 2004 Available online 13 January 2005

Keywords: Trypanosoma brucei; RNA interference; Mammalian host

RNA interference (RNAi) abrogates gene expression through transcription of double stranded RNA (dsRNA). It is mediated by a natural biological mechanism which triggers the transformation of these dsRNAs into shorter versions of 21–23 nt. These small interfering RNAs (siRNAs) then drive gene silencing by sequence specific mRNA degradation or by inhibition of protein synthesis. Endogenous RNAi generates active siRNAs from long natural dsRNAs or from microRNA (miRNA) hairpin structures [1–5]. RNAi can theoretically be used as a tool in all cell types possessing this endogenous pathway. Trypanosoma brucei falls into this category. The feasibility of this technique in trypanosomes was originally demonstrated by Gull and coworkers [6] and by Tschudi and co-workers [7]. Several groups (Wirtz and Clayton [8], Cross and co-workers [9], Englund and co-workers [10], Donelson and co-workers [11], Gull and co-workers [12] and Tschudi and co-workers [13]) have since engineered cell lines and plasmids allowing inducible dsRNA transcription from a fragment of interest [14,15]. T. brucei is a unicellular eukaryote belonging to the order kinetoplastidae. It is the agent of two major afflictions affecting the African continent, human sleeping sickness that kills an estimated 300,000 persons a year and nagana, which is the major obstacle to efficient cattle rearing throughout large regions of the African continent [http://www.who.org].

Abbreviations: dsRNA, double stranded RNA; miRNA, micro RNA; RNAi, RNA interference; siRNA, small interfering RNA ∗ Corresponding author. Tel.: +32 2 6509758; fax: +32 2 6509760. E-mail address: [email protected] (L. Vanhamme). 1 These authors contributed equally to this work. 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.12.009

Trypanosomes are a privileged model for the study of mechanisms involved in host–parasite interactions. These parasites escape their mammalian hosts defences through immunosuppressive activity [16], as well as antigenic variation [17,18], a regular change of their major surface antigen, the variant surface glycoprotein or VSG. In addition, some subspecies have also developed resistance to an innate trypanolytic factor [19]. While some aspects of these host–parasite interactions can be covered by studies in vitro, others, such as the analysis of trypanosome components on immunosuppressive activity, are better suited to in vivo studies. These studies would greatly benefit from the possibility of inducing RNAi in vivo. Two steps are required in order to obtain such a system. The currently available cell lines engineered for inducible RNAi are monomorphic strains, which have lost the ability to differentiate into stumpy forms. They kill laboratory rodents within a few days after injection. On the contrary, pleomorphic strains differentiate from proliferative slender forms into quiescent stumpy forms in the bloodstream, a process that allows the development of chronic infections. The first step towards in vivo RNAi would therefore be the creation of an equivalent pleomorphic cell line. In this paper, we address the second necessary step, the induction of RNAi in transgenic trypanosomes inside the mammalian host. To do so, systemic administration of tetracycline or an analogue is required. Tetracycline and its analogues have been successfully used in animals, in order to induce the cre recombinase in conditional knockout mice [20], and to induce protein over-expression in trypanosomes [21]. We performed RNAi-mediated knock down of two genes coding for general transcription factors: TbTFIIS1 (GeneDB accession number Tb11.02.2600) and TbXPD (GeneDB

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accession number Tb08.11J15.890). The former is one of two TFIIS homologues found in the T. brucei genome database (Walgraffe et al., in preparation), the latter is the T. brucei homologue of a subunit of the TFIIH transcription factor (Lecordier et al., submitted). PCR products (1–642 nt and 762–2160 nt of the TbTFIIS1 and TbXPD ORFs, respectively) were cloned in the pZJM vector [10] using the XhoI and HindIII restriction sites located between the two head-tohead inducible hybrid promoters. After linearization by NotI within the ribosomal spacer sequence, these DNA constructs were electroporated into the 13–90 bloodstream transgenic cell line expressing the T7 polymerase and the tetracycline sensitive repressor [9]. Transfected trypanosomes were selected in HMI-9 medium containing phleomycin. Selected populations were cloned on agar plates [22]. The expression of dsRNA was induced in culture by adding the tetracycline analogue doxycycline to 1 ␮g/ml into the culture medium. The genomic integration of the constructs was checked by Southern blot analysis (not shown), and the efficiency of induction was verified by Northern blot analysis (Fig. 1, panels A1 and B1). In both cases, the presence of doxycycline led to enhanced expression of dsRNA and concomitant decrease of the endogenous mRNA levels. This induction also led to reduction of protein expression to undetectable levels after

2 days (Fig. 1, panels A2 and B2). The growth of several clones of each transgenic cell line was monitored for several days in induced and control non-induced conditions. Similar results were obtained for all clones of 13–90pZJM TbTFIIS1 and of 13–90pZJM TbXPD. While the knock down of TFIIS1 only very mildly affected the cells, the TbXPD knock down led to cell death after 3 days (Fig. 1, panels A3 and B3, respectively). As a first approach to the use of RNAi in vivo, sets of six outbred NMRI mice (Charles River) were injected with 5 × 106 transgenic trypanosomes from two independent clones of 13–90pZJM TbTFIIS1 and of 13–90pZJM TbXPD. Half of the mice sets were given drinking water containing 1 mg/ml doxycycline that was changed every other day. The two clones of each transgenic line showed the same behaviour. In agreement with the results obtained in vitro, the parasitemia of mice injected with 13–90pZJM TbTFIIS1 was unaffected by the administration of doxycycline, killing the mice on day 3 after injection (Fig. 2A and B). In contrast, the 13–90pZJM TbXPD clones exhibited doxycycline dependant infection patterns. While untreated trypanosomes killed the mice on day 3 after injection (Fig. 2C), the doxycycline treated parasitemia grew to very mild levels on day 1 before falling to undetectable levels on the following days

Fig. 1. Knock down of TbTFIIS1 and TbXPD by RNAi. The 13–90 bloodstream cell line was transfected by pZJM TbTFIIS1 (panels A) or pZJM TbXPD (panels B) and cloned after selection. (A1 and B1) Northern blot analysis of RNA extracted from the cell lines during the growth curves before doxycycline addition (D0) or 1, 2 and 3 days (D1, D2 and D3) after doxycycline addition. The thin and open arrows, respectively, point at the mRNA and dsRNA. The upper panel shows the hybridization with the relevant probe while the lower one shows hybridization with a control histone H2B probe. (A2 and B2) Western blot analysis of total protein extracts from the cell lines during the growth curves before doxycycline addition (D0) and 1–3 days (D1–D3) after doxycycline addition. The arrowhead points at the specific band while the asterisk indicates a non-specific band of unknown nature inadvertently recognised by the antiserum and used as loading control. (A3 and B3) Growth curves of a clone from each cell line are shown in the absence () or presence (
) of doxycycline.

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Fig. 2. Parasitemia curves in mice watered with or without doxycycline, given on day 0 or 2 days before injection. The results are shown for one representative experiment out of four that were performed. Each curve and symbol represents an individual mouse. Large crosses indicate death. 5 × 106 bloodstream trypanosomes (13–90 strain) transfected with pZJMTFIIS (panels A and B) or pZJMXPD (panels C–E) were injected in each mouse. Mice were separated in groups of three, watered either with regular water (panels A and C), or water supplemented with doxycycline given on day 0 (panels B and D) or minus two (panel E).

(Fig. 2D). However, they relapsed between days 11 and 16 and killed the mice within 4 days (Fig. 2D). These results were reproduced twice. This suggests that administration of doxycycline in drinking water is sufficient to induce RNAi in vivo. The mild parasitemia observed on day 1 could be related to a lag period necessary either for the RNAi to affect growth or for doxycycline to reach its effective concentration in the blood. The relapse was observed in four out of six mice and three out of six mice, for each of the two clones. This suggests that some trypanosomes are able to escape the selective pressure of gene silencing imposed by RNAi. To address this question, we repeated the experiment with mice that were watered with doxycycline 48 h before injection (Fig. 2E). In this case, a mild parasitemia was still detected on day 1, but the mice remained negative thereafter for 25 days, and for an additional 15 days after stopping doxycycline treatment. This result was reproduced in a second independent experiment. This time lapse necessary for doxycycline to induce RNAi is also observed in culture. The period without selective pressure, occurring during the transfer of trypanosomes from culture where they are maintained under hygromycin/neomycin/phleomycin selection to

the moment doxycycline reaches effective concentrations in the host’s blood, could delay the kinetics of induction enough to allow RNAi revertants to appear. We addressed this possibility by physiological and genetic analysis of antibiotic resistance of two of the relapses. These trypanosomes were harvested and put in HMI-9 culture medium. They were found to have lost resistance to the antibiotics used for transgene selection. Southern blot analysis confirmed that these trypanosomes had lost part or all of the pZJM plasmid, since both TbXPD and phleoR transgenes were undetectable (results not shown). They further suggested that the relapsed populations were clonal. These observations emphasize the need to keep trypanosomes under permanent selective pressure in order to obtain a homogenous phenotype. Indeed, caution is recommended because in some cases RNAi induction imposes a negative pressure on cells. The ability of trypanosomes to perform genetic recombination could quickly lead to the appearance of relapsed trypanosomes and hide the effect of RNAi at the population level. Our results show that, provided that mice are prewatered with doxycycline, the system is strong enough to work in vivo. However, an ideal exper-

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imental protocol would involve a permanent selection for the three transgenes necessary for induction of RNAi. This would require watering the mice with geneticin, phleomycin and hygromycin. Although antitumoral activity [23] or selection [24,25] using two of these eukaryotic antibiotics in mice has been reported, the treatment was very short and the window of concentrations allowing selection without toxicity for mice was very narrow. Because of this toxicity, our attempts to select transgenic trypanosomes in mice, with a combination of these three antibiotics has so far failed (unpublished results). The rearrangement events observed in the relapsed trypanosomes occur with a probability in the order of 10−7 , as only 7 mice out of 12 injected with 5 × 106 13–90 pZJM TbXPD trypanosomes became positive in the presence of doxycycline. This conclusion was supported by the results of additional experiments in which two sets of four mice were injected, respectively, with 5 × 105 and 5 × 104 of these trypanosomes, in the presence of doxycycline. None of these mice exhibited parasites within the 30 days of monitoring. While RNAi emerges as a powerful tool in molecular biology, its application in vivo has been advocated. Pioneer experiments have demonstrated its effectiveness in antiviral therapy [26–28]. We have shown that an in vivo RNAi system could be used for studying host–parasite interactions, since our data establish the proof-of-principle for RNAi induction in trypanosomes grown in their hosts. This method should allow the study of genes/factors involved in mechanisms such as the rate of antigenic variation, resistance or sensitivity to lysis by human serum, or immunosuppression. These applications would also benefit from the development of pleomorphic cell strains suitable for RNAi, allowing the analysis of the immune response on a scale of weeks rather than days. On a longer term, it theoretically opens the door to treatment of trypanosomiasis by systemic delivery of miRNAs.

Acknowledgements The work in our laboratory was supported by the Interuniversity Attraction Poles Programme—Belgian Science Policy and a “credit aux chercheurs” given to L.V. by the Belgian National Fund for Scientific Research (FNRS). L.V. is Research Associate at the FNRS and D.W. and S.D. were supported by a FRIA fellowship. We thank Cecile Felu and Dr. Pierrick Uzureau for proofreading and valuable comments on the manuscript.

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