Suramin is one of the oldest drugs in use today. It is still the treatment of choice for the hemolymphatic stage of African sleeping sickness caused by Trypanosoma brucei rhodesiense, and it is also used for surra in camels caused by Trypanosoma evansi. Yet despite one hundred years of use, suramin's mode of action is not fully understood. Suramin is a polypharmacological molecule that inhibits diverse proteins. Here we demonstrate that a DNA helicase of the pontin/ruvB-like 1 family, termed T. brucei RuvBL1, is involved in suramin resistance in African trypanosomes. Bloodstream-form T. b. rhodesiense under long-term selection for suramin resistance acquired a homozygous point mutation, isoleucine-312 to valine, close to the ATP binding site of T. brucei RuvBL1. The introduction of this missense mutation, by reverse genetics, into drug-sensitive trypanosomes significantly decreased their sensitivity to suramin. Intriguingly, the corresponding residue of T. evansi RuvBL1 was found mutated in a suramin-resistant field isolate, in that case to a leucine. RuvBL1 (Tb927.4.1270) is predicted to build a heterohexameric complex with RuvBL2 (Tb927.4.2000). RNAi-mediated silencing of gene expression of either T. brucei RuvBL1 or RuvBL2 caused cell death within 72 h. At 36 h after induction of RNAi, bloodstream-form trypanosomes exhibited a cytokinesis defect resulting in the accumulation of cells with two nuclei and two or more kinetoplasts. Taken together, these data indicate that RuvBL1 DNA helicase is involved in suramin action in African trypanosomes.

• MeSH
• DNA-helikasy genetika   MeSH
• suramin farmakologie   terapeutické užití   MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma brucei rhodesiense genetika   MeSH
• Trypanosoma * genetika   MeSH
• trypanozomóza africká * farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Trypanosoma brucei parasites are the causative agents of African trypanosomiasis in humans, as well as surra, nagana, and dourine in animals. According to current widely used nomenclature, T. brucei is a group of five (sub)species, each causing a distinct disease and possessing unique genetic marker(s) or a combination thereof. However, minimal nuclear genome differences, sometimes accompanied by ongoing genetic exchange, robustly support polyphyly resulting from multiple independent origins of the (sub)species in nature. The ease of generating such (sub)species in the laboratory, as well as the case of overlapping hosts and disease symptoms, is incompatible with the current (sub)species paradigm, which implies a monophyletic origin. Here, we critically re-evaluate this concept, considering recent genome sequencing and experimental studies. We argue that ecotype should be used going forward as a significantly more accurate and appropriate designation.

• MeSH
• lidé MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma * genetika   MeSH
• trypanozomiáza * MeSH
• trypanozomóza africká * parazitologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

• MeSH
• citrátový cyklus účinky léků   MeSH
• glukosa metabolismus   MeSH
• hmyz - vektory parazitologie   MeSH
• moucha tse-tse účinky léků   parazitologie   MeSH
• oxidace-redukce účinky léků   MeSH
• prolin metabolismus   farmakologie   MeSH
• RNA interference fyziologie   MeSH
• Trypanosoma brucei brucei účinky léků   metabolismus   MeSH
• Trypanosoma účinky léků   metabolismus   MeSH
• trypanozomóza africká farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

• MeSH
• dobytek MeSH
• karboxypeptidasy metabolismus   MeSH
• kyseliny karboxylové metabolismus   MeSH
• léková rezistence MeSH
• myši MeSH
• parazitemie veterinární   MeSH
• prekurzory léčiv metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• sloučeniny boru metabolismus   MeSH
• trypanocidální látky metabolismus   MeSH
• Trypanosoma brucei brucei účinky léků   enzymologie   MeSH
• Trypanosoma congolense účinky léků   enzymologie   MeSH
• Trypanosoma vivax účinky léků   enzymologie   MeSH
• trypanozomóza africká farmakoterapie   parazitologie   veterinární   MeSH
• valin analogy a deriváty   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mutations in the Trypanosoma brucei aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.

• MeSH
• akvaporin 2 * chemie   genetika   metabolismus   MeSH
• akvaporiny chemie   genetika   metabolismus   MeSH
• léková rezistence účinky léků   genetika   MeSH
• melarsoprol farmakologie   MeSH
• mutace MeSH
• pentamidin farmakologie   MeSH
• trypanocidální látky farmakologie   MeSH
• Trypanosoma brucei brucei * účinky léků   genetika   metabolismus   MeSH
• trypanozomóza africká farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.

• MeSH
• akvaglyceroporiny chemie   metabolismus   MeSH
• léková rezistence * MeSH
• sekvence aminokyselin MeSH
• stabilita proteinů MeSH
• trypanocidální látky farmakologie   MeSH
• Trypanosoma brucei brucei * MeSH
• trypanozomóza africká parazitologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• farmakoterapie metody   MeSH
• lidé MeSH
• přenos infekční nemoci MeSH
• trypanozomóza africká * epidemiologie   farmakoterapie   přenos   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• novinové články MeSH
• zprávy MeSH
• Geografické názvy
• Kongo MeSH

• MeSH
• amébiáza * diagnóza   patofyziologie   přenos   terapie   MeSH
• leishmanióza kožní diagnóza   patofyziologie   přenos   terapie   MeSH
• leishmanióza viscerální diagnóza   patofyziologie   přenos   terapie   MeSH
• leishmanióza * diagnóza   patofyziologie   přenos   terapie   MeSH
• lidé MeSH
• moucha tse-tse MeSH
• toxoplazmóza * diagnóza   patofyziologie   přenos   terapie   MeSH
• trypanozomiáza * diagnóza   patofyziologie   přenos   terapie   MeSH
• trypanozomóza africká * diagnóza   patofyziologie   přenos   terapie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• přehledy MeSH

Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• azidy farmakologie   MeSH
• buněčné linie MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• mitochondriální DNA metabolismus   MeSH
• mitochondrie účinky léků   genetika   metabolismus   MeSH
• organofosforové sloučeniny chemie   farmakologie   MeSH
• protonové ATPasy metabolismus   MeSH
• RNA interference MeSH
• sukcinátdehydrogenasa metabolismus   MeSH
• trypanocidální látky farmakologie   MeSH
• Trypanosoma brucei brucei cytologie   účinky léků   růst a vývoj   MeSH
• trypanozomóza africká parazitologie   MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.

• MeSH
• duplikace genu MeSH
• flagella fyziologie   MeSH
• fylogeneze MeSH
• geneticky modifikované organismy MeSH
• myši inbrední C57BL MeSH
• proteinové domény MeSH
• protozoální proteiny chemie   genetika   imunologie   metabolismus   MeSH
• Trypanosoma brucei brucei genetika   patogenita   fyziologie   MeSH
• trypanozomóza africká parazitologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH