A molecular mechanism for eflornithine resistance in African trypanosomes

Abstract

Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.

Figure 1. Resistance in T. brucei brucei (A), Selection of eflornithine resistance in Trypanosoma brucei.

Black triangles and left hand y-axis show the eflornithine concentration in which the parasites grew. Bars and the right hand y-axis show molar IC₅₀ values at various stages of the selection process. One clone out of two is shown. (B), Treatment of mice infected with wildtype or eflornithine resistant parasites. Closed circles; untreated, open diamonds; 2% eflornithine, open squares; 5% eflornithine, closed triangles; pentamidine (2mg/ml).

Figure 2. Isobologram analysis of nifurtimox and eflornithine combination.

Closed circles show the IC₅₀ values of drugs alone. Open circles show the IC₅₀ values of the drug combinations.

Figure 3. Metabolomic analysis of eflornithine resistance and uptake.

(A), Relative abundance of polyamine metabolites in wildtype (WT) and eflornithine resistant (R) cell extracts. (B), Uptake of eflornithine in wildtype (filled bars) and resistant cells (hatched bars) over one hour. Stars indicate a significant difference at a 0.01 level between WT at time 0 and WT after 60 minutes. A hash indicates that R at time 0 and R after 60 minutes show no significant difference at a 0.05 level. (C), Eflornithine uptake in wildtype and resistant cells. ³H-eflornithine transported into wildtype (triangles) and resistant (circles) cells was measured over 30 minutes. Measurements are an average of four separate experiments, each with three internal replicates. Error bars are the standard error of the mean. Inset graph shows threonine uptake in the same cell lines. The y-axis shows nmol of threonine per 10⁷ cells. The x-axis shows the time in minutes.

Figure 4. Eflornithine (left) is a derivative of ornithine (right).

Figure 5. Cladogram of the amino acid transporters predicted to be in T. brucei and how amplification of wildtype and resistant cell PCR products of 17 amino acid transporters from the wildtype and resistant cell lines shows TbAAT6 to be absent.

Inset: Southern blotting showed the loss of TbAAT6 in resistant, but not wildtype cells. ODC (ornithine decarboxylase) and β-tubulin remained unchanged.

Figure 6. PCR analysis of the region of chromosome 8 housing the single copy TbAAT6 (black box) in T. brucei.

An area of DNA is missing including TbAAT6. The exact boundary of the missing area is unknown (represented by the dotted line). Genes are (left to right) Tb927.8.5410 (hypothetical), Tb927.8.5420 (hypothetical), Tb927.8.5430 (hypothetical), Tb927.8.5440 (Tb-24, a flagellar calcium-binding protein), Tb927.8.5450 (TbAAT6), Tb927.8.5460 (Tb-44 a flagellar calcium-binding protein), Tb927.8.5465 (Tb-24, a flagellar calcium-binding protein), Tb927.8.5470 (Tb-17 a flagellar calcium-binding protein), Tb927.8.5480 (hypothetical), Tb927.8.5490 (hypothetical). Not all of these genes were amplified as Tb17, Tb24 and Tb44 are repetitive throughout the genome.

Figure 7. RNAi and re-expression of TbAAT6.

(A), RNAi was induced for 12 days and the IC₅₀ value to eflornithine measured. Stars indicate significant difference at a 0.05 level compared to wildtype, whereas a hash indicates that RNAi and resistant lines show no significant difference. (B), The IC₅₀ value of a constitutive re-expressor of TbAAT6 put into the resistant line. Stars indicate a significant difference at a 0.05 level compared to resistant, whereas a hash indicates that wildtype and re-expressor show no significant difference. Wildtype 24.6±5.8 µM, Resistant 886±200 µM, Re-expressor 111±18 µM, RNAi 773±53 µM. IC₅₀ measurements were at least n = 5.