Transporters of Trypanosoma brucei-phylogeny, physiology, pharmacology.

Trypanosoma brucei comprise the causative agents of sleeping sickness, T. b. gambiense and T. b. rhodesiense, as well as the livestock-pathogenic T. b. brucei. The parasites are transmitted by the tsetse fly and occur exclusively in sub-Saharan Africa. T. brucei are not only lethal pathogens but have also become model organisms for molecular parasitology. We focus here on membrane transport proteins of T. brucei, their contribution to homeostasis and metabolism in the context of a parasitic lifestyle, and their pharmacological role as potential drug targets or routes of drug entry. Transporters and channels in the plasma membrane are attractive drug targets as they are accessible from the outside. Alternatively, they can be exploited to selectively deliver harmful substances into the trypanosome's interior. Both approaches require the targeted transporter to be essential: in the first case to kill the trypanosome, in the second case to prevent drug resistance due to loss of the transporter. By combining functional and phylogenetic analyses, we were mining the T. brucei predicted proteome for transporters of pharmacological significance. Here, we review recent progress in the identification of transporters of lipid precursors, amino acid permeases and ion channels in T. brucei.

Identification and characterization of the three members of the CLC family of anion transport proteins in Trypanosoma brucei.

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.

TbIRK is a signature sequence free potassium channel from Trypanosoma brucei locating to acidocalcisomes.

Potassium channels from prokaryotes and eukaryotes are usually recognized by a typical amino acid sequence TXTGY(F)G representing the ionic selectivity filter. Using a screening approach with ion channel family profiles but without the above motif, we identified a gene in Trypanosoma brucei that exhibits homology to inward rectifying potassium channels. We report here cloning of this ion channel named TbIRK. The protein is localized to acidocalcisomes in procyclic and in bloodstream form parasites. Functional properties of this channel were established after expression in Xenopus oocytes. Currents recorded in potassium medium show inward rectification and little time dependence. Surprisingly, this channel retains selectivity for potassium ions over sodium ions >7, in spite of the lack of the classical selectivity filter. The sequence GGYVG was predicted in silico to replace this filter motif. Point mutations of the corresponding glycine residues confirmed this at the functional level. The channel is inhibited by caesium ions but remains unaffected by barium ions up to 10 mM. TbIRK is to our knowledge the first potassium channel in T. brucei that localizes to the acidocalcisomes, organelles involved in the storage of phosphates and the response to osmotic stress that occurs during the life cycle of trypanosomes.

H+-dependent inorganic phosphate uptake in Trypanosoma brucei is influenced by myo-inositol transporter.

Trypanosoma brucei is an extracellular protozoan parasite that causes human African trypanosomiasis or "sleeping sickness". During the different phases of its life cycle, T. brucei depends on exogenous inorganic phosphate (Pi), but little is known about the transport of Pi in this organism. In the present study, we showed that the transport of 32Pi across the plasma membrane follows Michaelis-Menten kinetics and is modulated by pH variation, with higher activity at acidic pH. Bloodstream forms presented lower Pi transport in comparison to procyclic forms, that displayed an apparent K0.5 = 0.093 ± 0.008 mM. Additionally, FCCP (H+-ionophore), valinomycin (K+-ionophore) and SCH28080 (H+, K+-ATPase inhibitor) inhibited the Pi transport. Gene Tb11.02.3020, previously described to encode the parasite H+:myo-inositol transporter (TbHMIT), was hypothesized to be potentially involved in the H+:Pi cotransport because of its similarity with the Pho84 transporter described in S. cerevisiae and other trypanosomatids. Indeed, the RNAi mediated knockdown remarkably reduced TbHMIT gene expression, compromised cell growth and decreased Pi transport by half. In addition, Pi transport was inhibited when parasites were incubated in the presence of concentrations of myo-inositol that are above 300 µM. However, when expressed in Xenopus laevis oocytes, two-electrode voltage clamp experiments provided direct electrophysiological evidence that the protein encoded by TbHMIT is definitely a myo-inositol transporter that may be only marginally affected by the presence of Pi. These results confirmed the presence of a Pi carrier in T. brucei, similar to the H+-dependent inorganic phosphate system described in S. cerevisiae and other trypanosomatids. This transport system contributes to the acquisition of Pi and may be involved in the growth and survival of procyclic forms. In summary, this work presents the first description of a Pi transport system in T. brucei.

Flagellar membranes are rich in raft-forming phospholipids.

The observation that the membranes of flagella are enriched in sterols and sphingolipids has led to the hypothesis that flagella might be enriched in raft-forming lipids. However, a detailed lipidomic analysis of flagellar membranes is not available. Novel protocols to detach and isolate intact flagella from Trypanosoma brucei procyclic forms in combination with reverse-phase liquid chromatography high-resolution tandem mass spectrometry allowed us to determine the phospholipid composition of flagellar membranes relative to whole cells. Our analyses revealed that phosphatidylethanolamine, phosphatidylserine, ceramide and the sphingolipids inositol phosphorylceramide and sphingomyelin are enriched in flagella relative to whole cells. In contrast, phosphatidylcholine and phosphatidylinositol are strongly depleted in flagella. Within individual glycerophospholipid classes, we observed a preference for ether-type over diacyl-type molecular species in membranes of flagella. Our study provides direct evidence for a preferential presence of raft-forming phospholipids in flagellar membranes of T. brucei.

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 µM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Structure, function, and modulation of GABA(A) receptors.

The GABA(A) receptors are the major inhibitory neurotransmitter receptors in mammalian brain. Each isoform consists of five homologous or identical subunits surrounding a central chloride ion-selective channel gated by GABA. How many isoforms of the receptor exist is far from clear. GABA(A) receptors located in the postsynaptic membrane mediate neuronal inhibition that occurs in the millisecond time range; those located in the extrasynaptic membrane respond to ambient GABA and confer long-term inhibition. GABA(A) receptors are responsive to a wide variety of drugs, e.g. benzodiazepines, which are often used for their sedative/hypnotic and anxiolytic effects.

myo-Inositol uptake is essential for bulk inositol phospholipid but not glycosylphosphatidylinositol synthesis in Trypanosoma brucei.

myo-Inositol is an essential precursor for the production of inositol phosphates and inositol phospholipids in all eukaryotes. Intracellular myo-inositol is generated by de novo synthesis from glucose 6-phosphate or is provided from the environment via myo-inositol symporters. We show that in Trypanosoma brucei, the causative pathogen of human African sleeping sickness and nagana in domestic animals, myo-inositol is taken up via a specific proton-coupled electrogenic symport and that this transport is essential for parasite survival in culture. Down-regulation of the myo-inositol transporter using RNA interference inhibited uptake of myo-inositol and blocked the synthesis of the myo-inositol-containing phospholipids, phosphatidylinositol and inositol phosphorylceramide; in contrast, it had no effect on glycosylphosphatidylinositol production. This together with the unexpected localization of the myo-inositol transporter in both the plasma membrane and the Golgi demonstrate that metabolism of endogenous and exogenous myo-inositol in T. brucei is strictly segregated.