Genetic validation of aminoacyl-tRNA synthetases as drug targets in Trypanosoma brucei

Abstract

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

FIG 1

Reactions catalyzed by aaRR. AA, amino acid substrate; aaRS:AA-AMP, enzyme-bound aminoacyl-adenylate intermediate; AA-tRNA, amino acyl tRNA.

FIG 2

Effect of RNAi-induced knockdown of ThrRS in BSF T. brucei. Independent RNAi cell lines generated in BSF cells for ThrRS were analyzed for growth and for cell cycle progression following RNAi induction. (A) Growth curves for three clonal lines with or without Tet (data represent the means, and error bars represent the ranges). Cell numbers were monitored by hemocytometer, and cell number was calculated as the product of cell density and the total dilution. (B) Real-time qPCR analysis of uninduced cells and cells treated with Tet for 24 h and 48 h relative to TERT as the normalization standard. Analyses were run in triplicate, and the error bars represent the root mean square deviation (RMSD). (C) Genome analysis by microscopy: distributions of nuclei (N) and kinetoplasts (K). Cells were grown with or without Tet for 48 h, stained with DAPI, and examined by fluorescence microscopy. A total of 150 cells were counted per condition. (D and E) Flow analysis of cells minus (D) or plus (E) Tet for 48 h and stained with propidium iodide (PI). PI fluorescence (FL3-Area) is plotted versus cell count. The tallest peak in panel D represents G₀/G₁ (48%), the span between peaks represents the S phase (4.59%), and the second tallest peak represents G₀/M (27.6%). (F) Confocal imaging of representative cells before and after Tet induction (48 h).

FIG 3

Effect of RNAi-induced knockdown of ArgRS in BSF T. brucei. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C) Genome analysis by microscopy. (D and E) Fluorescence-activated cell sorter (FACS) analysis. (F) Confocal imaging. Details are as described for Fig. 2.

FIG 4

Effect of RNAi-induced knockdown of AsnRS in BSF T. brucei. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C) Genome analysis by microscopy. (D and E) Flow analysis. (F) Confocal imaging. Details are as described for Fig. 2.

FIG 5

Effect of RNAi-induced knockdown of PheRS in BSF T. brucei. (Top panels) Knockdown of PheRSα. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C) Genome analysis by microscopy. (D and E) Flow analysis. (F) Confocal imaging. (Bottom panels) Knockdown of PheRSβ. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C) Genome analysis by microscopy. (D and E) Flow analysis. (F) Confocal imaging. Details are as described for Fig. 2.

FIG 6

Effect of RNAi-induced knockdown of TrpRS-1 in BSF T. brucei. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C and D) FACS analysis. Details are as described for Fig. 2.

FIG 7

(Top panels) Effect of RNAi-induced knockdown of ValRS in BSF T. brucei. (A) Growth curves. Cell numbers were monitored by hemocytometer. (B) Real-time qPCR analysis. (C) Genome analysis by microscopy. (Bottom panel) Effect of RNAi-induced knockdown on GluRS in BSF T. brucei. (A) Growth curves. Cells were counted by particle counting, which also detects cell debris. Details are as described for Fig. 2.

FIG 8

Cell morphology and FACS analysis of wild-type SM T. brucei cells. (A) Genome analysis by microscopy. (B) Confocal imaging. (C and D) Flow analysis. Details are as described for Fig. 2.

FIG 9

Characterization of ThrRS expression and activity. (A) ThrRS activity assays for recombinant protein purified from E. coli. Data represent dependence of PO₄^-2 formation versus time. (B) Dependence of PO₄^-2 formation on ThrRS concentration. Two moles of PO₄^-2 are formed for every mole of tRNA^Thr that is acylated. (C) Dose response of T. brucei BSF growth and recombinant ThrRS activity versus concentration of borrelidin, a known ThrRS inhibitor. For inhibition of parasite growth, the EC₅₀ = 2.2 μM (1.8 to 2.5); for inhibition of ThrRS activity, the IC₅₀ = 0.066 μM (0.020 to 0.22), where values in parentheses represent the 95% confidence interval.

FIG 10

Subcellular localization analysis of T. brucei BSF expressing C-terminal V5-tagged ThrRS. Cells were fixed in 4% paraformaldehyde and stained with FITC-conjugated anti-V5 monoclonal antibodies. Mitochondria were stained with MitoTracker (MT), and DNA was stained with DAPI. Differential interference contrast (DIC) is shown. Images were merged as indicated.