Lynsey J. M. Wallace

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

ABSTRACT Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

The Trypanocide Diminazene Aceturate Is Accumulated Predominantly through the TbAT1 Purine Transporter: Additional Insights on Diamidine Resistance in African Trypanosomes

ABSTRACT Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [3H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a Km of 0.45 ± 0.11 μM, which was dose dependently inhibited by pentamidine and adenosine. The Ki values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [3H]diminazene and [3H]pentamidine. TbAT1-null mutants had lost almost all capacity for [3H]diminazene transport. However, this cell line still displayed a small but detectable rate of [3H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [3H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

Cloning, heterologous expression, and in situ characterization of the first high affinity nucleobase transporter from a protozoan.

While multiple nucleoside transporters, some of which can also transport nucleobases, have been cloned in recent years from many different organisms, no sequence information is available for the high affinity, nucleobase-selective transporters of metazoa, parazoa, or protozoa. We have identified a gene, TbNBT1, from Trypanosoma brucei brucei that encodes a 435-residue protein of the equilibrative nucleoside transporter superfamily. The gene was expressed in both the procyclic and bloodstream forms of the organism. Expression of TbNBT1 in a Saccharomyces cerevisiae strain lacking an endogenous purine transporter allowed growth on adenine as sole purine source and introduced a high affinity transport activity for adenine and hypoxanthine, with Km values of 2.1 ± 0.6 and 0.66 ± 0.22 μm, respectively, as well as high affinity for xanthine, guanine, guanosine, and allopurinol and moderate affinity for inosine. A transporter with an indistinguishable kinetic profile was identified in T. b. brucei procyclics and designated H4. RNA interference of TbNBT1 in procyclics reduced cognate mRNA levels by ∼80% and H4 transport activity by ∼90%. Expression of TbNBT1 in Xenopus oocytes further confirmed that this gene encodes the first high affinity nucleobase transporter from protozoa or animals to be identified at the molecular level.

Apicoplast Lipoic Acid Protein Ligase B Is Not Essential for Plasmodium falciparum

Lipoic acid (LA) is an essential cofactor of α-keto acid dehydrogenase complexes (KADHs) and the glycine cleavage system. In Plasmodium, LA is attached to the KADHs by organelle-specific lipoylation pathways. Biosynthesis of LA exclusively occurs in the apicoplast, comprising octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) and LA synthase. Salvage of LA is mitochondrial and scavenged LA is ligated to the KADHs by LA protein ligase 1 (LplA1). Both pathways are entirely independent, suggesting that both are likely to be essential for parasite survival. However, disruption of the LipB gene did not negatively affect parasite growth despite a drastic loss of LA (>90%). Surprisingly, the sole, apicoplast-located pyruvate dehydrogenase still showed lipoylation, suggesting that an alternative lipoylation pathway exists in this organelle. We provide evidence that this residual lipoylation is attributable to the dual targeted, functional lipoate protein ligase 2 (LplA2). Localisation studies show that LplA2 is present in both mitochondrion and apicoplast suggesting redundancy between the lipoic acid protein ligases in the erythrocytic stages of P. falciparum.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter.

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 ± 0.013 μM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3′-hydroxy, 5′-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (ΔG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Selective Transport of a New Class of Purine Antimetabolites by the Protozoan Parasite Trypanosoma brucei

Purine antimetabolites have been very successful therapeutic agents against a host of infectious diseases and malignancies. Success of the treatment relies as much on the efficient accumulation by the target cell or organism as it does on selective action on a vital biochemical pathway of the target cell. Here we compare the ability of a new class of tricyclic purine antimetabolites to interact with transporters from human erythrocytes or Trypanosoma brucei. We show that these compounds display a remarkable selectivity for the parasites transporters. The adenine analogue showed greater trypanocidal activity than the hypoxanthine or guanine analogues in vitro. #This work was funded by the Wellcome Trust.

Design and synthesis of a series of truncated neplanocin fleximers

In an effort to study the effects of flexibility on enzyme recognition and activity, we have developed several different series of flexible nucleoside analogues in which the purine base is split into its respective imidazole and pyrimidine components. The focus of this particular study was to synthesize the truncated neplanocin A fleximers to investigate their potential anti-protozoan activities by inhibition of S-adenosylhomocysteine hydrolase (SAHase). The three fleximers tested displayed poor anti-trypanocidal activities, with EC50 values around 200 μM. Further studies of the corresponding ribose fleximers, most closely related to the natural nucleoside substrates, revealed low affinity for the known T. brucei nucleoside transporters P1 and P2, which may be the reason for the lack of trypanocidal activity observed.