RESUMEN
Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.
Asunto(s)
Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Interferencia de ARN , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transducción de Señal , AMP Cíclico/metabolismo , Flagelos/metabolismoRESUMEN
AIM: The demand for bowel cancer screening (BCS) is expected to increase significantly within the next decade. Little is known about the intentions of the workforce required to meet this demand. The Joint Advisory Group on Gastrointestinal Endoscopy (JAG), the British Society of Gastroenterology (BSG) and Association of Coloproctology of Great Britain and Ireland (ACPGBI) developed the first BCS workforce survey. The aim was to assess endoscopist career intentions to aid in future workforce planning to meet the anticipated increase in BCS colonoscopy. METHODS: A survey was developed by JAG, BSG and ACPGBI and disseminated to consultant, clinical and trainee endoscopists between February and April 2020. Descriptive and comparative analyses were undertaken, supported with BCS data. RESULTS: There were 578 respondents. Screening consultants have a median of one programmed activity (PA) per week for screening, accounting for 40% of their current endoscopy workload. 38% of current screening consultants are considering giving up colonoscopy in the next 2-5 years. Retirement (58%) and pension issues (23%) are the principle reasons for this. Consultants would increase their screening PAs by 70% if able to do so. The top three activities that endoscopists would relinquish to further support screening were outpatient clinics, acute medical/surgical on call and ward cover. An extra 155 colonoscopists would be needed to fulfil increased demand and planned retirement at current PAs. CONCLUSION: This survey has identified a serious potential shortfall in screening colonoscopists in the next 5-10 years due to an ageing workforce and job plan pressures of aspirant BCS colonoscopists. We have outlined potential mitigations including reviewing job plans, improving workforce resources and supporting accreditation and training.
RESUMEN
Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.
Asunto(s)
Proteínas de Transporte de Nucleósidos/metabolismo , Proteínas Protozoarias/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Tricomoniasis/parasitología , Trichomonas vaginalis/metabolismo , Transporte Biológico , Clonación Molecular , Humanos , Cinética , Proteínas de Transporte de Nucleósidos/química , Proteínas de Transporte de Nucleósidos/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/genéticaRESUMEN
Trypanosoma congolense is a principal agent causing livestock trypanosomiasis in Africa, costing developing economies billions of dollars and undermining food security. Only the diamidine diminazene and the phenanthridine isometamidium are regularly used, and resistance is widespread but poorly understood. We induced stable diminazene resistance in T. congolense strain IL3000 in vitro. There was no cross-resistance with the phenanthridine drugs, melaminophenyl arsenicals, oxaborole trypanocides, or with diamidine trypanocides, except the close analogs DB829 and DB75. Fluorescence microscopy showed that accumulation of DB75 was inhibited by folate. Uptake of [3 H]-diminazene was slow with low affinity and partly but reciprocally inhibited by folate and by competing diamidines. Expression of T. congolense folate transporters in diminazene-resistant Trypanosoma brucei brucei significantly sensitized the cells to diminazene and DB829, but not to oxaborole AN7973. However, [3 H]-diminazene transport studies, whole-genome sequencing, and RNA-seq found no major changes in diminazene uptake, folate transporter sequence, or expression. Instead, all resistant clones displayed a moderate reduction in the mitochondrial membrane potential Ψm. We conclude that diminazene uptake in T. congolense proceed via multiple low affinity mechanisms including folate transporters; while resistance is associated with a reduction in Ψm it is unclear whether this is the primary cause of the resistance.
Asunto(s)
Diminazeno/farmacología , Potencial de la Membrana Mitocondrial/fisiología , Tripanocidas/farmacología , Trypanosoma congolense/efectos de los fármacos , Tripanosomiasis Africana/veterinaria , Tripanosomiasis Bovina/tratamiento farmacológico , Animales , Bovinos , Resistencia a Medicamentos/fisiología , Transportadores de Ácido Fólico/metabolismo , Fenantridinas/farmacología , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Tripanosomiasis Bovina/parasitologíaRESUMEN
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.
Asunto(s)
Acuaporina 2 , Pentamidina/farmacología , Trypanosoma brucei brucei , Animales , Acuaporina 2/química , Acuaporina 2/genética , Acuaporina 2/metabolismo , Acuaporinas/química , Acuaporinas/genética , Acuaporinas/metabolismo , Resistencia a Medicamentos/efectos de los fármacos , Resistencia a Medicamentos/genética , Melarsoprol/farmacología , Mutación , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológicoRESUMEN
Only a single drug against schistosomiasis is currently available and new drug development is urgently required but very few drug targets have been validated and characterised. However, regulatory systems including cyclic nucleotide metabolism are emerging as primary candidates for drug discovery. Here, we report the cloning of ten cyclic nucleotide phosphodiesterase (PDE) genes of S. mansoni, out of a total of 11 identified in its genome. We classify these PDEs by homology to human PDEs. Male worms displayed higher expression levels for all PDEs, in mature and juvenile worms, and schistosomula. Several functional complementation approaches were used to characterise these genes. We constructed a Trypanosoma brucei cell line in which expression of a cAMP-degrading PDE complements the deletion of TbrPDEB1/B2. Inhibitor screens of these cells expressing only either SmPDE4A, TbrPDEB1 or TbrPDEB2, identified highly potent inhibitors of the S. mansoni enzyme that elevated the cellular cAMP concentration. We further expressed most of the cloned SmPDEs in two pde1Δ/pde2Δ strains of Saccharomyces cerevisiae and some also in a specialised strain of Schizosacharomyces pombe. Five PDEs, SmPDE1, SmPDE4A, SmPDE8, SmPDE9A and SmPDE11 successfully complemented the S. cerevisiae strains, and SmPDE7var also complemented to a lesser degree, in liquid culture. SmPDE4A, SmPDE8 and SmPDE11 were further assessed in S. pombe for hydrolysis of cAMP and cGMP; SmPDE11 displayed considerable preferrence for cGMP over cAMP. These results and tools enable the pursuit of a rigorous drug discovery program based on inhibitors of S. mansoni PDEs.
Asunto(s)
Clonación Molecular , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas del Helminto/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Schistosoma mansoni/enzimología , Schistosoma mansoni/genética , Animales , Línea Celular , Eliminación de Gen , Perfilación de la Expresión Génica , Genoma de los Helmintos , Proteínas del Helminto/genética , Masculino , Ratones , Filogenia , Trypanosoma brucei brucei , LevadurasRESUMEN
BACKGROUND: Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, needs urgent alternative therapeutic options as the treatments currently available display severe limitations, mainly related to efficacy and toxicity. OBJECTIVES: As phosphodiesterases (PDEs) have been claimed as novel targets against T. cruzi, our aim was to evaluate the biological aspects of 12 new phthalazinone PDE inhibitors against different T. cruzi strains and parasite forms relevant for human infection. METHODS: In vitro trypanocidal activity of the inhibitors was assessed alone and in combination with benznidazole. Their effects on parasite ultrastructural and cAMP levels were determined. PDE mRNA levels from the different T. cruzi forms were measured by quantitative reverse transcription PCR. RESULTS: Five TcrPDEs were found to be expressed in all parasite stages. Four compounds displayed strong effects against intracellular amastigotes. Against bloodstream trypomastigotes (BTs), three were at least as potent as benznidazole. In vitro combination therapy with one of the most active inhibitors on both parasite forms (NPD-040) plus benznidazole demonstrated a quite synergistic profile (xΣ FICI = 0.58) against intracellular amastigotes but no interaction (xΣ FICI = 1.27) when BTs were assayed. BTs treated with NPD-040 presented disrupted Golgi apparatus, a swollen flagellar pocket and signs of autophagy. cAMP measurements of untreated parasites showed that amastigotes have higher ability to efflux this second messenger than BTs. NPD-001 and NPD-040 increase the intracellular cAMP content in both BTs and amastigotes, which is also released into the extracellular milieu. CONCLUSIONS: The findings demonstrate the potential of PDE inhibitors as anti-T. cruzi drug candidates.
Asunto(s)
Enfermedad de Chagas , Tripanocidas , Trypanosoma cruzi , Enfermedad de Chagas/tratamiento farmacológico , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfodiesterasa/uso terapéutico , Tripanocidas/farmacología , Tripanocidas/uso terapéuticoRESUMEN
Aim: Due to the urgent need for effective drugs to treat schistosomiasis that act through a known molecular mechanism of action, we focused on a target-based approach with the aim to discover inhibitors of a cyclic nucleotide phosphodiesterase from Schistosoma mansoni (SmPDE4A). Materials & methods: To discover new inhibitors of SmPDE4A homology models of the enzyme structure were constructed based on known human and protozoan homologs. The best two models were selected for subsequent virtual screening of our in-house chemical library. Results & conclusion: A total of 25 library compounds were selected for experimental confirmation as SmPDE4A inhibitors and after dose-response experiments, three top hits were identified. The results presented validate the virtual screening approach to identify new inhibitors for clinically relevant phosphodiesterases.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Descubrimiento de Drogas , Inhibidores de Fosfodiesterasa 4/farmacología , Schistosoma mansoni/enzimología , Esquistosomiasis/tratamiento farmacológico , Animales , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Inhibidores de Fosfodiesterasa 4/química , Esquistosomiasis/metabolismo , Relación Estructura-ActividadRESUMEN
Animal African trypanosomiasis (AAT) is a significant socioeconomic burden for sub-Saharan Africa because of its huge impact on livestock health. Existing therapies including those based on minor groove binders (MGBs), such as the diamidines, which have been used for decades, have now lost efficacy in some places because of the emergence of resistant parasites. Consequently, the need for new chemotherapies is urgent. Here, we describe a structurally distinct class of MGBs, Strathclyde MGBs (S-MGBs), which display excellent in vitro activities against the principal causative organisms of AAT: Trypanosoma congolense, and Trypanosoma vivax. We also show the cure of T. congolense-infected mice by a number of these compounds. In particular, we identify S-MGB-234, compound 7, as curative by using two applications of 50 mg/kg intraperitoneally. Crucially, we demonstrate that S-MGBs do not show cross-resistance with the current diamidine drugs and are not internalized via the transporters used by diamidines. This study demonstrates that S-MGBs have significant potential as novel therapeutic agents for AAT.
Asunto(s)
Tripanocidas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Ciclo Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Metabolómica , Ratones , Pentamidina/química , Pentamidina/farmacología , Relación Estructura-Actividad , Tripanocidas/química , Tripanocidas/metabolismo , Tripanocidas/farmacología , Trypanosoma congolense/efectos de los fármacos , Trypanosoma congolense/crecimiento & desarrollo , Trypanosoma congolense/metabolismoRESUMEN
We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100⯵M, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (nâ¯=â¯72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro 'hit' NPD-0274â¯at 20â¯mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10â¯mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs.
Asunto(s)
Antihelmínticos/farmacología , Imidazoles/farmacología , Schistosoma mansoni/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Animales , Antihelmínticos/química , Descubrimiento de Drogas , Fibroblastos/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Imidazoles/química , Masculino , Ratones , Recuento de Huevos de Parásitos , Praziquantel/farmacologíaRESUMEN
The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.
Asunto(s)
Leishmaniasis/tratamiento farmacológico , Leishmaniasis/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Animales , Antiprotozoarios/uso terapéutico , AMP Cíclico/metabolismo , Leishmania major/efectos de los fármacos , Leishmania major/enzimología , Leishmania major/patogenicidad , Masculino , Ratones , Ratones Endogámicos BALB C , Inhibidores de Fosfodiesterasa/uso terapéuticoRESUMEN
Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Inhibidores de Fosfodiesterasa/química , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/química , Amidas/química , Amidas/farmacología , Dominio Catalítico , Concentración 50 Inhibidora , Modelos Moleculares , Terapia Molecular Dirigida , Proteínas Protozoarias/química , Relación Estructura-ActividadRESUMEN
The transport of nucleobases and nucleosides in protozoan parasites is known to be performed by Equilibrative Nucleoside Transporter (ENT) family members, including the extensively studied P1 and P2 nucleoside transporters of T. brucei bloodstream forms. Studies with P2 knockout parasites suggested the existence of as yet uncharacterised purine transport mechanisms in these cells. Here, we deleted several ENT genes, in addition to P2, including an array comprising three genes encoding for high-affinity broad-selectivity nucleobase transporters - the longest multi-gene locus deletion in T. brucei to date. It was verified that none of them appreciably contributed to the transport of hypoxanthine in bloodstream forms grown axenically in HMI-9 medium, which was mainly performed by a previously not described hypoxanthine-specific transporter (HXT1) with a Km of 22⯱â¯1.7⯵M and Vmax of 0.49⯱â¯0.06â¯pmol(107â¯cells)-1â¯s-1. The uptake of adenine was also assessed in the knockout cells and was performed by a highly specific adenine transporter (ADET1) with a Km of 573⯱â¯62â¯nM and Vmax of 0.23⯱â¯0.06â¯pmol(107â¯cells)-1â¯s-1. Neither HXT1 nor ADET1 displayed any affinity for other natural purines or pyrimidines and could not be completely inhibited by hypoxanthine or adenine analogues. These carriers may be the final pieces in the substantial transporter array trypanosomes can employ to fine-tune the uptake of purines from diverse environments during their life cycles, and may be encoded by genes other than those of the ENT family.
Asunto(s)
Adenina/metabolismo , Hipoxantina/metabolismo , Proteínas de Transporte de Nucleobases/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/metabolismo , Medios de Cultivo/química , Eliminación de Gen , Cinética , Proteínas de Transporte de Nucleobases/genética , Especificidad por Sustrato , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
BACKGROUND: Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine. METHODOLOGY/PRINCIPAL FINDINGS: Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15), being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA) and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance. CONCLUSIONS/SIGNIFICANCE: Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which allows loss of kDNA and results in a reduction of the mitochondrial membrane potential.
Asunto(s)
Resistencia a Medicamentos/genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Fenantridinas/farmacología , ATPasas de Translocación de Protón/genética , Proteínas Protozoarias/genética , Tripanocidas/farmacología , Animales , Benzamidinas/farmacología , ADN de Cinetoplasto/genética , Femenino , Ratones , Ratones Endogámicos ICR , Mutación , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
Trypanosoma brucei rhodesiense is one of the causative agents of human sleeping sickness, a fatal disease that is transmitted by tsetse flies and restricted to Sub-Saharan Africa. Here we investigate two independent lines of T. b. rhodesiense that have been selected with the drugs melarsoprol and pentamidine over the course of 2 years, until they exhibited stable cross-resistance to an unprecedented degree. We apply comparative genomics and transcriptomics to identify the underlying mutations. Only few mutations have become fixed during selection. Three genes were affected by mutations in both lines: the aminopurine transporter AT1, the aquaporin AQP2, and the RNA-binding protein UBP1. The melarsoprol-selected line carried a large deletion including the adenosine transporter gene AT1, whereas the pentamidine-selected line carried a heterozygous point mutation in AT1, G430R, which rendered the transporter non-functional. Both resistant lines had lost AQP2, and both lines carried the same point mutation, R131L, in the RNA-binding motif of UBP1. The finding that concomitant deletion of the known resistance genes AT1 and AQP2 in T. b. brucei failed to phenocopy the high levels of resistance of the T. b. rhodesiense mutants indicated a possible role of UBP1 in melarsoprol-pentamidine cross-resistance. However, homozygous in situ expression of UBP1-Leu(131) in T. b. brucei did not affect the sensitivity to melarsoprol or pentamidine.
Asunto(s)
Resistencia a Medicamentos/genética , Genoma de Protozoos , Trypanosoma brucei rhodesiense/genética , Secuencia de Aminoácidos , Acuaporinas/genética , Acuaporinas/metabolismo , Hibridación Genómica Comparativa , ADN Protozoario/química , ADN Protozoario/aislamiento & purificación , ADN Protozoario/metabolismo , Heterocigoto , Humanos , Masculino , Melarsoprol/farmacología , Proteínas de Transporte de Nucleósidos/genética , Proteínas de Transporte de Nucleósidos/metabolismo , Pruebas de Sensibilidad Parasitaria , Pentamidina/farmacología , Fenotipo , Polimorfismo de Nucleótido Simple , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia , Tripanocidas/farmacología , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/aislamiento & purificación , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/parasitologíaRESUMEN
Aquaglyceroporin-2 is a known determinant of melarsoprol-pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2-AQP3 tandem locus was described from melarsoprol-pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2-aqp3 null T. b. brucei does not. This proves that AQP2-AQP3 chimerization is the cause of melarsoprol-pentamidine cross-resistance in the T. b. gambiense isolates.
Asunto(s)
Acuaporina 2/metabolismo , Acuaporina 3/metabolismo , Melarsoprol/farmacología , Pentamidina/farmacología , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/genética , Acuaporina 2/genética , Acuaporina 3/genética , Resistencia a Medicamentos , Regulación de la Expresión Génica/fisiología , Tripanocidas/farmacologíaRESUMEN
Drug resistance in pathogenic protozoa is very often caused by changes to the 'transportome' of the parasites. In Trypanosoma brucei, several transporters have been implicated in uptake of the main classes of drugs, diamidines and melaminophenyl arsenicals. The resistance mechanism had been thought to be due to loss of a transporter known to carry both types of agents: the aminopurine transporter P2, encoded by the gene TbAT1. However, although loss of P2 activity is well-documented as the cause of resistance to the veterinary diamidine diminazene aceturate (DA; Berenil(®)), cross-resistance between the human-use arsenical melarsoprol and the diamidine pentamidine (melarsoprol/pentamidine cross resistance, MPXR) is the result of loss of a separate high affinity pentamidine transporter (HAPT1). A genome-wide RNAi library screen for resistance to pentamidine, published in 2012, gave the key to the genetic identity of HAPT1 by linking the phenomenon to a locus that contains the closely related T. brucei aquaglyceroporin genes TbAQP2 and TbAQP3. Further analysis determined that knockdown of only one pore, TbAQP2, produced the MPXR phenotype. TbAQP2 is an unconventional aquaglyceroporin with unique residues in the "selectivity region" of the pore, and it was found that in several MPXR lab strains the WT gene was either absent or replaced by a chimeric protein, recombined with parts of TbAQP3. Importantly, wild-type AQP2 was also absent in field isolates of T. b. gambiense, correlating with the outcome of melarsoprol treatment. Expression of a wild-type copy of TbAQP2 in even the most resistant strain completely reversed MPXR and re-introduced HAPT1 function and transport kinetics. Expression of TbAQP2 in Leishmania mexicana introduced a pentamidine transport activity indistinguishable from HAPT1. Although TbAQP2 has been shown to function as a classical aquaglyceroporin it is now clear that it is also a high affinity drug transporter, HAPT1. We discuss here a possible structural rationale for this remarkable ability.
RESUMEN
The Trypanosoma brucei aminopurine transporter P2/TbAT1 has long been implicated in the transport of, and resistance to, the diamidine and melaminophenyl arsenical classes of drugs that form the backbone of the pharmacopoeia against African trypanosomiasis. Genetic alterations including deletions and single nucleotide polymorphisms (SNPs) have been observed in numerous strains and clinical isolates. Here, we systematically investigate each reported mutation and assess their effects on transporter function after expression in a tbat1(-/-) T. brucei line. Out of a set of six reported SNPs from a reported 'resistance allele', none significantly impaired sensitivity to pentamidine, diminazene or melarsoprol, relative to the TbAT1-WT allele, although several combinations, and the deletion of the codon for residue F316, resulted in highly significant impairment. These combinations of SNPs, and ΔF316, also strongly impaired the uptake of [(3)H]-adenosine and [(3)H]-diminazene, identical to the tbat1(-/-) control. The TbAT1 protein model predicted that residues F19, D140 and F316 interact with the substrate of the transporter. Mutation of D140 to alanine resulted in an inactive transporter, whereas the mutation F19A produced a transporter with a slightly increased affinity for [(3)H]-diminazene but reduced the uptake rate. The results presented here validate earlier hypotheses of drug binding motifs for TbAT1.
Asunto(s)
Modelos Moleculares , Proteínas de Transporte de Nucleósidos/química , Proteínas de Transporte de Nucleósidos/genética , Tripanocidas/farmacología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Alelos , Diminazeno/farmacología , Resistencia a Múltiples Medicamentos/genética , Cinética , Melarsoprol/farmacología , Mutación , Proteínas de Transporte de Nucleósidos/metabolismo , Pruebas de Sensibilidad Parasitaria , Pentamidina/farmacología , Polimorfismo de Nucleótido Simple , Dominios y Motivos de Interacción de Proteínas , Trypanosoma brucei brucei/químicaRESUMEN
OBJECTIVES: Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. METHODS: The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. RESULTS: All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. CONCLUSIONS: TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter.