title: RNA-interference silencing of the adenosine transporter-1 gene in Trypanosoma evansi confers resistance to diminazene aceturate.
abstract: Drug resistance of trypanosomes is now a problem, but its underlying mechanisms are not fully understood. Cellular uptake of the major trypanocidal drugs is thought to occur through an adenosine transporter. The adenosine transporter-1 gene, TbAT1, encoding a P2-like nucleoside transporter has previously been cloned from Trypanosoma brucei brucei, and when expressed in yeast, it showed very similar substrate specificity to the P2-nucleoside transporter, but could not transport diamidines (pentamidine and diminazene). We have cloned and sequenced a similar gene (TevAT1) from Trypanosoma evansi and found it to have 99.7% identity to the TbAT1 gene. To elucidate the role of the TevAT1 gene on diamidine trypanocidal effect, we genetically engineered T. evansi for conditional knock-out of the TevAT1 gene by RNA interference (RNAi). Induction of the RNAi resulted in 10-fold depletion of TevAT1 mRNA, with concomitantly significant resistance to diminazene aceturate (berenil). The induced parasites propagated normally and attained peak cell density at an in vitro concentration of berenil, 5.5-fold higher than the IC(100) of the wild-type. TevAT1 knock-out had no effect on the trypanocidal activity of suramin and antrycide, but conferred some resistance to samorin. Our findings validate the significance of the TevAT1 adenosine transporter-1 gene in mediating the trypanocidal effect of diamidines in T. evansi. Further, we show for the first time that RNAi gene silencing in T. evansi can be induced using plasmids designed for T. brucei. We also demonstrate the usefulness of real-time PCR in rapidly quantifying mRNA levels in trypanosomes.
authors.affiliation: Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
authors.forename: William H
authors.initials: WH
authors.lastname: Witola
authors.forename: Noboru
authors.initials: N
authors.lastname: Inoue
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ohashi
authors.forename: Misao
authors.initials: M
authors.lastname: Onuma
mesh_terms: D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry; D003670:Deer; D004133:Diminazene / Q000031:analogs & derivatives* / Q000494:pharmacology* / Q000627:therapeutic use; D004351:Drug Resistance; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D033703:Nucleoside Transport Proteins; D021261:Parasitic Sensitivity Tests; D010617:Phenanthridines / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011806:Quinolinium Compounds / Q000494:pharmacology; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D013498:Suramin / Q000494:pharmacology; D014344:Trypanocidal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D014345:Trypanosoma / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014352:Trypanosomiasis / Q000188:drug therapy* / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan; D026901:Membrane Transport Proteins; D033703:Nucleoside Transport Proteins; D010617:Phenanthridines; D011806:Quinolinium Compounds; D012333:RNA, Messenger; D014344:Trypanocidal Agents; C444719:adenosine transporter; D013498:Suramin; C000702:isometamidium chloride; C005707:quinapyramine; C003915:diminazene aceturate; D004133:Diminazene