Tests of cytoplasmic RNA interference (RNAi) and construction of a tetracycline-inducible T7 promoter system in Trypanosoma cruzi

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Abstract

The technique of RNA interference (RNAi) is exceedingly useful for knocking down the expression of a specific mRNA in African trypanosomes and other organisms for the purpose of examining the function of its gene. However, when we attempted to apply RNAi in the Latin American trypanosome, Trypanosoma cruzi, to diminish expression of mRNA encoding the surface protein amastin, we found that the amastin double-stranded RNA (dsRNA) was not efficiently degraded in either epimastigotes or amastigotes, and the level of amastin mRNA remained unchanged. We generated a strain of T. cruzi CL-Brener in which the T7 promoter and tetracycline operator could be used to maximize tetracycline-regulated dsRNA synthesis and constructed plasmids that direct dsRNA against four different T. cruzi endogenous genes (encoding β-tubulin, GP72 (flagellar adhesion protein), ribosomal protein P0 and amastin) and an exogenously added gene (GFP; green fluorescent protein). After either stable or transient transfection of these plasmids into T. cruzi, the expected RNAi phenotype was not observed for any of the five genes, although the T. cruzi β-tubulin RNAi plasmid did give the expected FAT cell phenotype in the African trypanosome, Trypanosoma brucei. These data indicate that, similar to Leishmania, T. cruzi lacks one or more components necessary for the RNAi pathway and that these components will need to be engineered into T. cruzi, or compensated for, before RNAi can be used to study gene function in this organism.

Introduction

The phenomenon of RNA interference (RNAi) was initially described in the nematode Caenorhabditis elegans [1] and subsequently shown in the same organism to act via the presence of double-stranded (ds) RNA molecules [2]. RNAi is now known to occur in a variety of organisms, including two species of African trypanosomes, Trypanosoma brucei [3] and Trypanosoma congolense [4]. It has become a powerful experimental technique for depleting cells of a specific mRNA in the cytoplasm that has, in turn, triggered a revolution in the study of gene function in those cells and organisms where it can be applied. Much effort has been expended in elucidating the molecular mechanisms by which RNAi functions (for a recent review, see [5]). Briefly, long dsRNA molecules are cleaved by an RNase-III activity (called Dicer in some organisms) to short interfering dsRNAs (siRNAs) of 21–22-nucleotides that have 5′-phosphates and single-strand extensions of two or three nucleotides with 3′-hydroxyl groups. These duplex siRNAs are unwound by a multiprotein RNA-inducing silencing complex (RISC) and the antisense strand guides the RISC to a complementary mRNA sequence, which becomes a target for endonucleolytic cleavage. The antisense strand and the mRNA appear to form a duplex and the mRNA is cleaved in the middle of the duplex, about 10 nucleotides from the 5′ end of the siRNA [5], [6].

The Latin American trypanosome, Trypanosoma cruzi, is the causative agent of Chagas disease, a significant human health problem in Latin America. The T. cruzi life cycle has three main developmental stages. Epimastigotes multiply extracellularly in the midgut of the reduviid bug vector and migrate to the hindgut, where they differentiate into non-dividing metacyclic trypomastigotes that are excreted. These infective trypomastigotes pass through mucous membranes and skin cuts of a mammalian host where they invade a number of cell types and differentiate into amastigotes, which multiply intracellularly in the cytoplasm of the parasitized cell. Amastigotes transform into trypomastigotes and enter the circulatory system when the host cells lyze. These non-dividing bloodstream trypomastigotes invade new host cells and can be transmitted to a reduviid bug in its blood meal to complete the life cycle.

We previously identified and characterized a T. cruzi protein called amastin, whose mRNA is 50–70-fold more abundant in amastigotes than epimastigotes [7]. A homologue of T. cruzi amastin occurs in Leishmania, which is, likewise, much more abundant in Leishmania’s intracellular amastigote stage than in its extracellular promastigote stage [8]. Nascent T. cruzi amastin is a glycoprotein of 174 amino acids that has four distinct hydrophobic regions of 20–30 amino acids, one at each terminus and two at internal locations. Its prominent location on the surface of amastigotes suggests that one or more of the hydrophobic regions is a trans-membrane segment, although this possibility has not been demonstrated and the function of amastin remains unknown.

Since RNAi had been shown to act in the two African trypanosome species, we anticipated it might be possible to use this same approach to diminish amastin mRNA in T. cruzi and detect a phenotype during the T. cruzi life cycle that would provide clues about amastin’s function. In particular, we thought that since amastin mRNA’s abundance is low in epimastigotes and high in amastigotes, RNAi against amastin mRNA might have little or no effect in epimastigotes, but would have a deleterious effect on either epimastigote differentiation to amastigotes or survival of amastigotes in mammalian host cells. We found, however, that the amastin dsRNA had no detectable effect on either epimastigotes or amastigotes, and that it was not substantively degraded in either cell type. This finding led us to investigate whether RNAi against β-tubulin (which yields a distinctive FAT (rounded cell) phenotype in African trypanosomes [3]) or against GP72 (which yields a detached flagellum in African trypanosomes [9]) would give a similar phenotype in T. cruzi. In addition, we also constructed a strain of T. cruzi that expresses T7 RNA polymerase (T7RNP) and the tetracycline repressor (TetR) so we could utilize (i) the T7RNP promoter (PT7) to increase synthesis of the dsRNA and (ii) the tetracycline operator (Otet) to control this synthesis. However, under none of these circumstances did we detect evidence for an RNAi phenotype, leading us to conclude that T. cruzi lacks one or more of the endogenous components required for functional RNAi.

Section snippets

Parasites

The epimastigote form of T. cruzi CL-Brener strain [10] was grown in liver digested neutralized tryptose (LDNT) medium supplemented with 10% fetal calf serum (FCS) at 28 °C, or in M199 medium plus 10% LDNT supplemented with FCS [11]. The procyclic T. brucei cell line 29-13 [12], a gift from G.A.M. Cross, Rockefeller University, was maintained at 28 °C in Cunningham’s SM medium plus 10% FCS.

Plasmid constructions

To generate the plasmid pTREX/AMAdsRNA (Fig. 1A), a PCR-amplified fragment containing a 305-bp segment of

Stable expression of amastin dsRNA has no effect on T. cruzi epimastigotes or amastigotes

As a first step to test whether amastin (AMA) dsRNA might confer a readily detectable phenotype on either T. cruzi epimastigotes or amastigotes, plasmid pTREX/AMAdsRNA was constructed (Fig. 1A). This plasmid contains a T. cruzi rRNA gene promoter (PrRNA) followed by two opposing copies of a 305-bp segment of the 522-bp AMA coding region that are separated by a 746-bp spacer. The circular form of this plasmid was electroporated into T. cruzi CL-Brener epimastigotes, transfectants were selected

Discussion

The initial goal of this work was to develop RNAi as a tool in T. cruzi for potentially determining the biological role of AMA, a membrane glycoprotein encoded by multiple genes whose mRNA abundance increases 50–70-fold as epimastigotes differentiate to amastigotes [7]. A plasmid was introduced into T. cruzi from which AMA dsRNA was synthesized as a “stem-loop” hairpin configuration (Fig. 1A). Northern blots revealed that the dsRNA was indeed synthesized, but not substantively degraded, as

Acknowledgements

This work was supported by NIH grants AI40591 and TW00958. W.D.R. and S.M.R.T. received further support from the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq) in Brazil.

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