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A molecular mechanism for eflornithine resistance in African trypanosomes.

Vincent IM, Creek D, Watson DG, Kamleh MA, Woods DJ, Wong PE, Burchmore RJ, Barrett MP - PLoS Pathog. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Glasgow, Glasgow, UK.

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.

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Eflornithine (left) is a derivative of ornithine (right).
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ppat-1001204-g004: Eflornithine (left) is a derivative of ornithine (right).

Mentions: These data indicated a transporter phenotype, as seen previously in selection of resistance to melamine based arsenicals [13] and diamidines [14], [16], [20], [21]. As eflornithine is an amino acid analogue (Fig. 4), we hypothesised loss of an amino acid transporter. To test this, members of the amino acid permease gene family (Fig. 5) in the T. brucei genome [22] were systematically amplified from both wildtype and each of the two independently selected resistant lines. In each of the independently selected lines only one single copy amino acid transporter gene, TbAAT6 (Tb927.8.5450), was shown to be absent (Fig. 5). PCR analysis indicated a deletion of this, and surrounding genes, from both resistant lines (DFMOR1, Fig. 6, R2 not shown). This result indicated the possibility that the TbAAT6 gene could play a role in eflornithine's entry into T. brucei and that its loss was responsible for drug resistance. The gene was amplifiable at day 34 (Fig. 1A), but by day 50 (Fig. 1A) was no longer amplifiable.


A molecular mechanism for eflornithine resistance in African trypanosomes.

Vincent IM, Creek D, Watson DG, Kamleh MA, Woods DJ, Wong PE, Burchmore RJ, Barrett MP - PLoS Pathog. (2010)

Eflornithine (left) is a derivative of ornithine (right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991269&req=5

ppat-1001204-g004: Eflornithine (left) is a derivative of ornithine (right).
Mentions: These data indicated a transporter phenotype, as seen previously in selection of resistance to melamine based arsenicals [13] and diamidines [14], [16], [20], [21]. As eflornithine is an amino acid analogue (Fig. 4), we hypothesised loss of an amino acid transporter. To test this, members of the amino acid permease gene family (Fig. 5) in the T. brucei genome [22] were systematically amplified from both wildtype and each of the two independently selected resistant lines. In each of the independently selected lines only one single copy amino acid transporter gene, TbAAT6 (Tb927.8.5450), was shown to be absent (Fig. 5). PCR analysis indicated a deletion of this, and surrounding genes, from both resistant lines (DFMOR1, Fig. 6, R2 not shown). This result indicated the possibility that the TbAAT6 gene could play a role in eflornithine's entry into T. brucei and that its loss was responsible for drug resistance. The gene was amplifiable at day 34 (Fig. 1A), but by day 50 (Fig. 1A) was no longer amplifiable.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: University of Glasgow, Glasgow, UK.

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.

Show MeSH
Related in: MedlinePlus