scholarly journals Multiple Genetic Mechanisms Lead to Loss of Functional TbAT1 Expression in Drug-Resistant Trypanosomes

2009 ◽  
Vol 9 (2) ◽  
pp. 336-343 ◽  
Author(s):  
Mhairi L. Stewart ◽  
Richard J. S. Burchmore ◽  
Caroline Clucas ◽  
Christiane Hertz-Fowler ◽  
Karen Brooks ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Drug Resistant ◽  
Diminazene Aceturate ◽  
Content Type ◽  
African Trypanosomes ◽  
Genomic Location ◽  
Adenosine Uptake ◽  
Resistant Lines ◽  
The Status ◽  
Genetic Mechanisms

ABSTRACT The P2 aminopurine transporter, encoded by TbAT1 in African trypanosomes in the Trypanosoma brucei group, carries melaminophenyl arsenical and diamidine drugs into these parasites. Loss of this transporter contributes to drug resistance. We identified the genomic location of TbAT1 to be in the subtelomeric region of chromosome 5 and determined the status of the TbAT1 gene in two trypanosome lines selected for resistance to the melaminophenyl arsenical, melarsamine hydrochloride (Cymelarsan), and in a Trypanosoma equiperdum clone selected for resistance to the diamidine, diminazene aceturate. In the Trypanosoma brucei gambiense STIB 386 melarsamine hydrochloride-resistant line, TbAT1 is deleted, while in the Trypanosoma brucei brucei STIB 247 melarsamine hydrochloride-resistant and T. equiperdum diminazene-resistant lines, TbAT1 is present, but expression at the RNA level is no longer detectable. Further characterization of TbAT1 in T. equiperdum revealed that a loss of heterozygosity at the TbAT1 locus accompanied loss of expression and that P2-mediated uptake of [3H]diminazene is lost in drug-resistant T. equiperdum. Adenine-inhibitable adenosine uptake is still detectable in a ΔTbat1 T. b. brucei mutant, although at a greatly reduced capacity compared to that of the wild type, indicating that an additional adenine-inhibitable adenosine permease, distinct from P2, is present in these cells.

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

2004 ◽  
Vol 48 (5) ◽  
pp. 1515-1519 ◽  
Author(s):  
Harry P. de Koning ◽  
Laura F. Anderson ◽  
Mhairi Stewart ◽  
Richard J. S. Burchmore ◽  
Lynsey J. M. Wallace ◽  
...  
Keyword(s):  
Cell Line ◽  
Trypanosoma Brucei ◽  
Domestic Animals ◽  
Trypanosoma Brucei Brucei ◽  
Diminazene Aceturate ◽  
Severe Problem ◽  
African Trypanosomes ◽  
Detectable Rate ◽  
Almost All ◽  
Null Mutants

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.

scholarly journals Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

2020 ◽  
Author(s):  
Kariuki Ndung’u ◽  
Grace Adira Murilla ◽  
John Kibuthu Thuita ◽  
Geoffrey Njuguna Ngae ◽  
Joanna Eseri Auma ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Post Infection ◽  
Drug Resistant ◽  
Weight Changes ◽  
Trypanosoma Brucei Rhodesiense ◽  
Diminazene Aceturate ◽  
Reference Drug ◽  
Western Kenya ◽  
Biotechnology Research ◽  
Cure Rates

AbstractWe assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n=10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0-15, (b) acute: 16-30, (c) sub-acute: 31-45 and (d) chronic: 46-60 dpi. Other virulence biomarkers identified included: prepatent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B) pentamidine, diminazene aceturate and suramin, using mice groups (n= 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. At least 80% cure rates of all the test isolates was achieved with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr isolates from a single HAT focus and confirms that these variations are not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

Uptake of purine bases and nucleosides in African trypanosomes

Parasitology ◽  
1980 ◽  
Vol 81 (2) ◽  
pp. 383-393 ◽  
Author(s):  
Dinah M. James ◽  
G. V. R. Born
Keyword(s):  
Trypanosoma Brucei ◽  
Trypanosoma Congolense ◽  
The Other ◽  
African Trypanosomes ◽  
Purine Bases ◽  
Marked Inhibition ◽  
Adenosine Uptake

SUMMARYUptake of radioactively labelled purine bases and nucleosides by suspensions of Trypanosoma brucei and Trypanosoma congolense in bicine buffer was determined at 37 °C. With T. brucei, the rate of uptake of adenosine was much greater than that of the other compounds tested, the uptake of which decreased in the order adenine, inosine, guanosine and hypoxanthine. With T. brucei, adenosine uptake increased with concentration in a manner suggesting two mechanisms, one with high and the other with low affinity for adenosine. The uptake of adenine increased with concentration only up to about 1·5 µM while the uptake of guanosine increased little with concentration and that of inosine and hypoxanthine not at all. In both species adenosine strongly inhibited the uptake of both of the other nucleosides and of both purine bases. In T. brucei, guanosine and inosine caused small increases in adenosine uptake which was, however, inhibited by them in T. congolense. In T. brucei, each of the purine bases adenine and hypoxanthine inhibited its own uptake maximally but that of each other less effectively. Hypoxanthine was more effective than adenine in inhibiting the uptake of the nucleosides guanosine and inosine, but neither base effected marked inhibition of adenosine uptake. The uptake of adenosine by T. brucei was inhibited by dipyridamole and its analogue, compound RA–233, strongly at 100µM and slightly at 10 µM. The other dipyridamole analogues, VK–744 and VK–774, were ineffective.

scholarly journals Trypanocidal Activity of 8-Methyl-5′-{[(Z)-4-Aminobut-2-enyl]-(Methylamino)}Adenosine (Genz-644131), an Adenosylmethionine Decarboxylase Inhibitor

2009 ◽  
Vol 53 (8) ◽  
pp. 3269-3272 ◽  
Author(s):  
Cyrus J. Bacchi ◽  
Robert H. Barker ◽  
Aixa Rodriguez ◽  
Bradford Hirth ◽  
Donna Rattendi ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Dose Range ◽  
System Model ◽  
Drug Resistant ◽  
Trypanosoma Brucei Rhodesiense ◽  
Trypanosoma Brucei Brucei ◽  
African Trypanosomes ◽  
Adenosylmethionine Decarboxylase ◽  
Daily Dose ◽  
Purine Ring

ABSTRACT Genzyme 644131, 8-methyl-5′-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine, is an analog of the enzyme activated S-adenosylmethionine decarboxylase (AdoMetDC) inhibitor and the trypanocidal agent MDL-7381, 5-{[(Z)-4-aminobut-2-enyl](methylamino)}adenosine. The analog differs from the parent in having an 8-methyl group on the purine ring that bestows favorable pharmacokinetic, biochemical, and trypanocidal activities. The compound was curative in acute Trypanosoma brucei brucei and drug-resistant Trypanosoma brucei rhodesiense model infections, with single-dose activity in the 1- to 5-mg/kg/day daily dose range for 4 days against T. brucei brucei and 25- to 50-mg/kg twice-daily dosing against T. brucei rhodesiense infections. The compound was not curative in the TREU 667 central nervous system model infection but cleared blood parasitemia and extended time to recrudescence in several groups. This study shows that AdoMetDC remains an attractive chemotherapeutic target in African trypanosomes and that chemical changes in AdoMetDC inhibitors can produce more favorable drug characteristics than the lead compound.

The relationship between duration of infection with Trypanosoma brucei in mice and the efficacy of chemotherapy

Parasitology ◽  
1977 ◽  
Vol 75 (2) ◽  
pp. 143-153 ◽  
Author(s):  
F. W. Jennings ◽  
D. D. Whitelaw ◽  
G. M. Urquhart
Keyword(s):  
Drug Resistance ◽  
Drug Treatment ◽  
Trypanosoma Brucei ◽  
Post Infection ◽  
Drug Resistant ◽  
Diminazene Aceturate ◽  
Duration Of Infection ◽  
Permanent Cure ◽  
Dose Levels ◽  
The Relationship

Relapse of infection after drug treatment of trypanosome infections under conditions precluding re-infection has usually been ascribed to drug resistance on the part of the parasite or to under-dosage of the drug. With Trypanosoma brucei infection in mice we have obtained evidence of another type of relapse. In infections resulting from the inoculation of 1 × 105 trypanosomes, derived from a stabilate T. brucei TREU 667, treatment with diminazene aceturate (Berenil) at 40 mg/kg at either 3 or 7 days after infection elicited a permanent cure. If, however, treatment was delayed later than 14 days after infection, then all the mice relapsed. These relapses generally occurred between 20 and 50 days after treatment, but some mice remained aparasitaemic for up to 60 days. The relapsed infections were apparently not due to the survival of ‘drug-resistant’ trypanosomes, as infections derived from a stabilate isolated from a relapsed Berenil-treated mouse were also permanently cured with Berenil if treated 3 days after infection; however, if treatment was delayed until 21 days post-infection, all the mice relapsed. The cause of relapse was not related to the number of parasites inoculated, as infection resulting from initial inocula of 1 × 105 to 1 × 108 trypanosomes were all cured if treated at 3 days after infection, and all eventually relapsed if treatment was delayed until day 21. This type of relapse phenomenon was not confined to T. brucei TREU 667 but also occurred with 5 other stabi-lates of T. brucei after Berenil treatment. Treatment of T. brucei TREU 667 infections with Ethidium and Prothidium at dose levels of 7.5 and 10 mg/kg respectively was also followed by relapse if treatment was delayed for 3 weeks after infection. The possible causes of relapse under these conditions, and its implications in the study of the natural disease, are discussed.

scholarly journals Positional Dynamics and Glycosomal Recruitment of Developmental Regulators during Trypanosome Differentiation

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Balázs Szöőr ◽  
Dorina V. Simon ◽  
Federico Rojas ◽  
Julie Young ◽  
Derrick R. Robinson ◽  
...  
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Tyrosine Phosphatase ◽  
Tsetse Fly ◽  
Sub Saharan Africa ◽  
Metabolic Adaptation ◽  
Tsetse Flies ◽  
Content Type ◽  
African Trypanosomes ◽  
Sub Saharan

ABSTRACT Glycosomes are peroxisome-related organelles that compartmentalize the glycolytic enzymes in kinetoplastid parasites. These organelles are developmentally regulated in their number and composition, allowing metabolic adaptation to the parasite’s needs in the blood of mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates differentiation between parasite developmental forms, comprising a tyrosine phosphatase, Trypanosoma brucei PTP1 (TbPTP1), which dephosphorylates and inhibits a serine threonine phosphatase, TbPIP39, which promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to glycosomes during differentiation from bloodstream “stumpy” forms to procyclic forms. Detailed microscopy and live-cell imaging during the synchronous transition between life cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site closely associated with TbVAP, which defines the flagellar pocket endoplasmic reticulum. TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of stumpy-enriched mRNAs. This site provides a molecular node for the interaction between TbPTP1 and TbPIP39. Within 30 min of the initiation of differentiation, TbPIP39 relocates to glycosomes, whereas TbPTP1 disperses to the cytosol. Overall, the study identifies a “stumpy regulatory nexus” (STuRN) that coordinates the molecular components of life cycle signaling and glycosomal development during transmission of Trypanosoma brucei. IMPORTANCE African trypanosomes are parasites of sub-Saharan Africa responsible for both human and animal disease. The parasites are transmitted by tsetse flies, and completion of their life cycle involves progression through several development steps. The initiation of differentiation between blood and tsetse fly forms is signaled by a phosphatase cascade, ultimately trafficked into peroxisome-related organelles called glycosomes that are unique to this group of organisms. Glycosomes undergo substantial remodeling of their composition and function during the differentiation step, but how this is regulated is not understood. Here we identify a cytological site where the signaling molecules controlling differentiation converge before the dispersal of one of them into glycosomes. In combination, the study provides the first insight into the spatial coordination of signaling pathway components in trypanosomes as they undergo cell-type differentiation.

scholarly journals Activity of Aromathecins against African Trypanosomes

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Nathaniel P. Nenortas ◽  
Maris A. Cinelli ◽  
Andrew E. Morrell ◽  
Mark Cushman ◽  
Theresa A. Shapiro
Keyword(s):  
Trypanosoma Brucei ◽  
Sleeping Sickness ◽  
Dna Intercalation ◽  
African Sleeping Sickness ◽  
Content Type ◽  
African Trypanosomes ◽  
Antitrypanosomal Activity ◽  
Topoisomerase Ib

ABSTRACT African sleeping sickness is responsible for thousands of deaths annually, and new therapeutics are needed. This study evaluated aromathecins, experimental inhibitors of mammalian topoisomerase IB, against Trypanosoma brucei African trypanosomes. The compounds had selectively toxic antiparasitic potency, in situ poisoning activity against the phylogenetically unique topoisomerase in these parasites, and a representative compound intercalated into DNA with micromolar affinity. DNA intercalation and topoisomerase poisoning may contribute to the antitrypanosomal activity of aromathecins.

scholarly journals Antitrypanosomal Activity of a New Triazine Derivative, SIPI 1029, In Vitro and in Model Infections

1998 ◽  
Vol 42 (10) ◽  
pp. 2718-2721 ◽  
Author(s):  
Cyrus J. Bacchi ◽  
Marcus Vargas ◽  
Donna Rattendi ◽  
Burt Goldberg ◽  
Weicheng Zhou
Keyword(s):  
Trypanosoma Brucei ◽  
Model Systems ◽  
In Vivo Model ◽  
Trypanosoma Brucei Rhodesiense ◽  
Diminazene Aceturate ◽  
African Trypanosomes ◽  
Cure Rates ◽  
Laboratory Models

ABSTRACT A recently developed diaminotriazine derivative [O,O′-bis(1,2-dihydro-2,2-tetramethylene-4,6-diamino-S-triazin-1-yl)-1,6-hexanediol dihydrochloride; T-46; SIPI 1029] was examined for activity against African trypanosomes in in vitro and in vivo model systems. In vitro, SIPI 1029 was 50% inhibitory for growth of bloodstream trypomastigotes of four strains of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense at 0.15 to 2.15 nM (50% inhibitory concentrations). In in vivo mouse laboratory models of T. b. rhodesiense clinical isolate infections, SIPI 1029 was curative for 12 of 13 isolates at ≤10 mg/kg of body weight/day for 3 days. In eight infections, a single dose was ≥60% curative, and in six of these, a dose of ≤5 mg/kg was sufficient for ≥60% cure rates. A number of these isolates were resistant to the standard trypanocide melarsoprol (Arsobal) and/or the diamidines diminazene aceturate (Berenil) and pentamidine. SIPI 1029 was also curative in combination withdl-α-difluoromethylornithine (Ornidyl) in a T. b. brucei central nervous system model infection. Some evidence of toxicity was found in dosage regimens of 10 mg/kg/day for 2 or 3 days in which deaths were observed in 6 of 65 animals given this dosage regimen. The activity of SIPI 1029 in this study indicates that this class of compounds (diaminotriazines) should be explored as leads for new human and veterinary trypanocides.

scholarly journals Potent Antitrypanosomal Activities of 3-Aminosteroids against African Trypanosomes: Investigation of Cellular Effects and of Cross-Resistance with Existing Drugs

Molecules ◽  
2019 ◽  
Vol 24 (2) ◽  
pp. 268 ◽  
Author(s):  
Charles Nnadi ◽  
Godwin Ebiloma ◽  
Jennifer Black ◽  
Ngozi Nwodo ◽  
Leandro Lemgruber ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cross Resistance ◽  
Veterinary Drug ◽  
Drug Resistant ◽  
M Cell ◽  
Diminazene Aceturate ◽  
Cell Viability Assay ◽  
African Trypanosomes ◽  
Mitochondrial Membrane Depolarization

Treatment of animal African trypanosomiasis (AAT) requires urgent need for safe, potent and affordable drugs and this has necessitated this study. We investigated the trypanocidal activities and mode of action of selected 3-aminosteroids against Trypanosoma brucei brucei. The in vitro activity of selected compounds of this series against T. congolense (Savannah-type, IL3000), T. b. brucei (bloodstream trypomastigote, Lister strain 427 wild-type (427WT)) and various multi-drug resistant cell lines was assessed using a resazurin-based cell viability assay. Studies on mode of antitrypanosomal activity of some selected 3-aminosteroids against Tbb 427WT were also carried out. The tested compounds mostly showed moderate-to-low in vitro activities and low selectivity to mammalian cells. Interestingly, a certain aminosteroid, holarrhetine (10, IC50 = 0.045 ± 0.03 µM), was 2 times more potent against T. congolense than the standard veterinary drug, diminazene aceturate, and 10 times more potent than the control trypanocide, pentamidine, and displayed an excellent in vitro selectivity index of 2130 over L6 myoblasts. All multi-drug resistant strains of T. b. brucei tested were not significantly cross-resistant with the purified compounds. The growth pattern of Tbb 427WT on long and limited exposure time revealed gradual but irrecoverable growth arrest at ≥ IC50 concentrations of 3-aminosteroids. Trypanocidal action was not associated with membrane permeabilization of trypanosome cells but instead with mitochondrial membrane depolarization, reduced adenosine triphosphate (ATP) levels and G2/M cell cycle arrest which appear to be the result of mitochondrial accumulation of the aminosteroids. These findings provided insights for further development of this new and promising class of trypanocide against African trypanosomes.

scholarly journals Differential virulence of Trypanosoma brucei rhodesiense isolates does not influence the outcome of treatment with anti-trypanosomal drugs in the mouse model

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0229060
Author(s):  
Kariuki Ndung’u ◽  
Grace Adira Murilla ◽  
John Kibuthu Thuita ◽  
Geoffrey Njuguna Ngae ◽  
Joanna Eseri Auma ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Post Infection ◽  
Drug Resistant ◽  
Weight Changes ◽  
Cure Rate ◽  
Trypanosoma Brucei Rhodesiense ◽  
Diminazene Aceturate ◽  
Reference Drug ◽  
Western Kenya ◽  
Biotechnology Research

We assessed the virulence and anti-trypanosomal drug sensitivity patterns of Trypanosoma brucei rhodesiense (Tbr) isolates in the Kenya Agricultural and Livestock Research Organization-Biotechnology Research Institute (KALRO-BioRI) cryobank. Specifically, the study focused on Tbr clones originally isolated from the western Kenya/eastern Uganda focus of human African Trypanosomiasis (HAT). Twelve (12) Tbr clones were assessed for virulence using groups(n = 10) of Swiss White Mice monitored for 60 days post infection (dpi). Based on survival time, four classes of virulence were identified: (a) very-acute: 0–15, (b) acute: 16–30, (c) sub-acute: 31–45 and (d) chronic: 46–60 dpi. Other virulence biomarkers identified included: pre-patent period (pp), parasitaemia progression, packed cell volume (PCV) and body weight changes. The test Tbr clones together with KALRO-BioRi reference drug-resistant and drug sensitive isolates were then tested for sensitivity to melarsoprol (mel B), pentamidine, diminazene aceturate and suramin, using mice groups (n = 5) treated with single doses of each drug at 24 hours post infection. Our results showed that the clones were distributed among four classes of virulence as follows: 3/12 (very-acute), 3/12 (acute), 2/12 (sub-acute) and 4/12 (chronic) isolates. Differences in survivorship, parasitaemia progression and PCV were significant (P<0.001) and correlated. The isolate considered to be drug resistant at KALRO-BioRI, KETRI 2538, was confirmed to be resistant to melarsoprol, pentamidine and diminazene aceturate but it was not resistant to suramin. A cure rate of at least 80% was achieved for all test isolates with melarsoprol (1mg/Kg and 20 mg/kg), pentamidine (5 and 20 mg/kg), diminazene aceturate (5 mg/kg) and suramin (5 mg/kg) indicating that the isolates were not resistant to any of the drugs despite the differences in virulence. This study provides evidence of variations in virulence of Tbr clones from a single HAT focus and confirms that this variations is not a significant determinant of isolate sensitivity to anti-trypanosomal drugs.

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