Validation of spermidine synthase as a drug target in African trypanosomes

Abstract

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.

Figure 1. Polyamine biosynthetic pathway in trypanosomatids

In one arm of the pathway, putrescine is synthesized from arginine via ornithine by the enzymes arginase (1) and ODC (2), the latter being the target for the trypanocidal agent DFMO. In the other arm of the pathway, methionine is converted into SAM (S-adenosylmethionine) and on to dcSAM by the activities of SAM synthetase (3) and SAMdc (4) respectively. SpSyn (5) then catalyses the formation of spermidine from putrescine and dcSAM. In trypanosomatids, spermidine is required for biosynthesis of the unique thiol trypanothione.

Figure 2. Sequence analysis of TbSpSyn

Alignment of SpSyn sequences from T. brucei (accession number XP_827124), T. cruzi (XP_811725), L. major (CAC44919), Arabidopsis thaliana (CAB61614), human (AAA36633), Saccharomyces cerevisiae (yeast) (AAC17191) and Plasmodium falciparum (AAN35885). Residues in common with the T. brucei sequence are highlighted in light grey; dashes represent gaps in the sequence to optimize alignment. Amino acids that form the characteristic PAPT family signature motif (Prosite documentation PDOC01033) are indicated by ‘#’. Residues that are believed to interact with the substrates dcSAM (His⁷⁴, Asp⁹⁸, Asp¹¹⁸, Asp¹⁴⁹, Gly¹⁵⁰, Asp¹⁶⁸ and Pro¹⁷⁵) (dark grey) and putrescine (Trp¹⁹, Gln⁶⁴, Tyr⁷³, Asp¹⁶⁸, Thr¹⁶⁹, Asp¹⁷¹, Glu²⁰³, Leu²⁰⁷, Asn²⁰⁸, Gln²⁰⁹, Tyr²³⁸, Pro²³⁹ and Ile²⁴³) (black) are highlighted [48,49].

Figure 3. Biochemical properties of TbSpSyn

(A) SDS/10% PAGE gel stained with Coomassie Brilliant Blue. Lane 1, crude extract loaded on to an Ni-NTA column and flow through (lane 2). The column was washed extensively with 10 mM imidazole (lane 3) and the recombinant protein eluted with 200 mM imidazole (lane 4). Markers (M) are given in kilodaltons. The 37 kDa band (indicated) corresponds to recombinant TbSpSyn. (B) Interaction of TbSpSyn with putrescine. TbSpSyn activity was monitored by following the production of spermidine from putrescine (12.5-1000 μM) and dcSAM (10 μM) by using 100 μg of TbSpSyn. (C) Interaction of TbSpSyn with dcSAM. TbSpSyn activity was monitored by following the production of spermidine from putrescine (1000 μM) and dcSAM (0.05-10 μM) in the presence of 100 μg of TbSpSyn. In (B, C), reactions were dansylated and spermidine levels were determined by HPLC coupled with a fluorescence detector. Activity is expressed as nmol of spermidine formed · min^-1 · mg^-1.

Figure 4. Spermidine biosynthesis is essential for BSF T. brucei

(A) The growth of tetracycline-treated (+ tetracycline) and non-treated (- tetracycline) BSF T. brucei was followed in the parental (SMB) and RNAi cell lines. The growth defect could be complemented by expressing the LmSpSyn gene in the RNAi cell line (RNAi + Comp). The results are expressed as cumulative cell number · ml^-1. (B) Down-regulation of gene expression by RNAi. Blots containing 15 μg of T. brucei total RNA from tetracycline-induced (96 h) (+) and non-induced (-) BSF cell lines were hybridized with radiolabelled probes as indicated. n.d., Not done. Equal loading was judged by ethidium bromide staining of rRNA.

Figure 5. BSF T. brucei lack a spermidine uptake capacity

BSF T. brucei or T. cruzi epimastigotes (2 × 10⁸ parasites · ml^-1 per assay) were incubated with [¹⁴C]spermidine for the times indicated. Cells were prepared and uptake of the radiolabel was measured by scintillation counting (see the Experimental section). Results are the means ± S.D. for three experiments.