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.

• Klíčová slova
• Drug target, Drug-resistance, RuvB-like 1 DNA helicase, Suramin, Trypanosoma brucei, Trypanosoma evansi,
• 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
• Názvy látek
• DNA-helikasy MeSH
• suramin MeSH

Control of protein levels is vital to cellular homeostasis, for maintaining a steady state, to coordinate changes during differentiation and other roles. In African trypanosomes surface proteins contribute to immune evasion, drug sensitivity and environmental sensing. The trypanosome surface is dominated by the GPI-anchored variant surface glycoprotein, but additional GPI-anchored and trans-membrane domain proteins are present with known roles as nutrient receptors and signal transducers. The evolutionarily conserved deubiquitinase orthologs of Usp7 and Vdu1 in trypanosomes modulate abundance of many surface proteins, including the invariant surface glycoproteins, which have roles in immune evasion and drug sensitivity. Here we identify multiple trypanosome Skp1 paralogs and specifically a divergent paralog SkpZ. Affinity isolation and LCMSMS indicates that SkpZ forms a heterotrimeric complex with TbUsp7 and TbTpr86, a tetratricopeptide-repeat protein. Silencing SkpZ decreases TbUsp7 and TbTpr86 abundance, confirming a direct association. Further, SkpZ knockdown decreases the abundance of multiple trans-membrane domain (TMD) proteins but increases GPI-anchored surface protein levels. Hence, a heterotrimeric complex of TbTpr86, TbUsp7 and SkpZ (TUSK) regulates expression levels of a significant cohort of trypanosome surface proteins mediating coordination between TMD and GPI-anchored protein expression levels.

• Klíčová slova
• cullin complex, deubiquitinase, drug sensitivity, surface proteins, trafficking, trypanosomes, ubiquitylation,
• Publikační typ
• časopisecké články MeSH

The surface proteins of parasitic protozoa mediate functions essential to survival within a host, including nutrient accumulation, environmental sensing and immune evasion. Several receptors involved in nutrient uptake and defence from the innate immune response have been described in African trypanosomes and, together with antigenic variation, contribute towards persistence within vertebrate hosts. Significantly, a superfamily of invariant surface glycoproteins (ISGs) populates the trypanosome surface, one of which, ISG75, is implicated in uptake of the century-old drug suramin. By CRISPR/Cas9 knockout and biophysical analysis, we show here that ISG75 directly binds suramin and mediates uptake of additional naphthol-related compounds, making ISG75 a conduit for entry of at least one structural class of trypanocidal compounds. However, ISG75 null cells present only modest attenuation of suramin sensitivity, have unaltered viability in vivo and in vitro and no alteration to suramin-invoked proteome responses. While ISG75 is demonstrated as a valid suramin cell entry pathway, we suggest the presence of additional mechanisms for suramin accumulation, further demonstrating the complexity of trypanosomatid drug interactions and potential for evolution of resistance.

• Klíčová slova
• CRISPR/Cas9, drug accumulation, drug metabolism, invariant surface glycoprotein, suramin, trypanosome, xenobiotics,
• Publikační typ
• časopisecké články MeSH

Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events and pass a quality control system to protect the cytoplasm from the translation of aberrant proteins. Many of these events are highly conserved across eukaryotes, reflecting their ancient origin, but significant deviation from a canonical pathway as described from animals and fungi has emerged in the trypanosomatids. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may also reveal additional, previously unsuspected, mRNA export pathways.

• Klíčová slova
• eukaryogenesis, mRNA export, nuclear pore complex, polycistronic transcription, trans-splicing, trypanosomes,
• MeSH
• aktivní transport - buněčné jádro genetika   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• messenger RNA genetika   MeSH
• RNA * genetika   metabolismus   MeSH
• Trypanosoma * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• messenger RNA MeSH
• RNA * MeSH

Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.

• Klíčová slova
• Aquaglyceroporin, Trypanosoma brucei, drug resistance, membrane trafficking, pentamidine, sleeping sickness,
• MeSH
• akvaglyceroporiny genetika   MeSH
• léková rezistence genetika   MeSH
• protozoální proteiny genetika   MeSH
• trypanocidální látky farmakologie   MeSH
• Trypanosoma účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• akvaglyceroporiny MeSH
• protozoální proteiny MeSH
• trypanocidální látky MeSH

Salivarian trypanosomes comprise a group of extracellular anthroponotic and zoonotic parasites. The only sustainable method for global control of these infection is through vaccination of livestock animals. Despite multiple reports describing promising laboratory results, no single field-applicable solution has been successful so far. Conventionally, vaccine research focusses mostly on exposed immunogenic antigens, or the structural molecular knowledge of surface exposed invariant immunogens. Unfortunately, extracellular parasites (or parasites with extracellular life stages) have devised efficient defense systems against host antibody attacks, so they can deal with the mammalian humoral immune response. In the case of trypanosomes, it appears that these mechanisms have been perfected, leading to vaccine failure in natural hosts. Here, we provide two examples of potential vaccine candidates that, despite being immunogenic and accessible to the immune system, failed to induce a functionally protective memory response. First, trypanosomal enolase was tested as a vaccine candidate, as it was recently characterized as a highly conserved enzyme that is readily recognized during infection by the host antibody response. Secondly, we re-addressed a vaccine approach towards the Invariant Surface Glycoprotein ISG75, and showed that despite being highly immunogenic, trypanosomes can avoid anti-ISG75 mediated parasitemia control.

• Klíčová slova
• ISG75, enolase, trypanosomosis, vaccination,
• Publikační typ
• časopisecké články 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
• Názvy látek
• AN11736 MeSH Prohlížeč
• karboxypeptidasy MeSH
• kyseliny karboxylové MeSH
• prekurzory léčiv MeSH
• protozoální proteiny MeSH
• serine carboxypeptidase MeSH Prohlížeč
• sloučeniny boru MeSH
• trypanocidální látky MeSH
• valin 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.

African sleeping sickness is a potentially deadly illness caused by the parasite Trypanosoma brucei. The disease is treatable, but many of the current treatments are old and are becoming increasingly ineffective. For instance, resistance is growing against pentamidine, a drug used in the early stages in the disease, as well as against melarsoprol, which is deployed when the infection has progressed to the brain. Usually, cases resistant to pentamidine are also resistant to melarsoprol, but it is still unclear why, as the drugs are chemically unrelated. Studies have shown that changes in a water channel called aquaglyceroporin 2 (TbAQP2) contribute to drug resistance in African sleeping sickness; this suggests that it plays a role in allowing drugs to kill the parasite. This molecular ‘drain pipe’ extends through the surface of T. brucei, and should allow only water and a molecule called glycerol in and out of the cell. In particular, the channel should be too narrow to allow pentamidine or melarsoprol to pass through. One possibility is that, in T. brucei, the TbAQP2 channel is abnormally wide compared to other members of its family. Alternatively, pentamidine and melarsoprol may only bind to TbAQP2, and then ‘hitch a ride’ when the protein is taken into the parasite as part of the natural cycle of surface protein replacement. Alghamdi et al. aimed to tease out these hypotheses. Computer models of the structure of the protein were paired with engineered changes in the key areas of the channel to show that, in T. brucei, TbAQP2 provides a much broader gateway into the cell than observed for similar proteins. In addition, genetic analysis showed that this version of TbAQP2 has been actively selected for during the evolution process of T. brucei. This suggests that the parasite somehow benefits from this wider aquaglyceroporin variant. This is a new resistance mechanism, and it is possible that aquaglyceroporins are also larger than expected in other infectious microbes. The work by Alghamdi et al. therefore provides insight into how other germs may become resistant to drugs.

• Klíčová slova
• Trypanosoma brucei, aquaporin, biochemistry, chemical biology, drug resistance, drug transport, infectious disease, melarsoprol, microbiology, pentamidine,
• 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
• Názvy látek
• akvaporin 2 * MeSH
• akvaporiny MeSH
• melarsoprol MeSH
• pentamidin MeSH
• trypanocidální látky 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
• Názvy látek
• akvaglyceroporiny MeSH
• trypanocidální látky MeSH

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

• Klíčová slova
• Trypanosoma brucei, differentiation, drug action, drug mechanisms, energy homeostasis, glycosomes, metabolomics, parasite metabolism, polypharmacology, proteomics, sleeping sickness, suramin, trypanosome,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• energetický metabolismus účinky léků   MeSH
• flagella účinky léků   metabolismus   ultrastruktura   MeSH
• glykolýza účinky léků   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metabolom účinky léků   MeSH
• mikrotělíska účinky léků   metabolismus   ultrastruktura   MeSH
• mitochondrie účinky léků   metabolismus   ultrastruktura   MeSH
• molekulární modely MeSH
• prolin metabolismus   MeSH
• proteom metabolismus   MeSH
• protonové ATPasy metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• suramin farmakologie   MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• kyselina pyrohroznová MeSH
• prolin MeSH
• proteom MeSH
• protonové ATPasy MeSH
• protozoální proteiny MeSH
• suramin MeSH