Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche

Campagnaro, Gustavo Daniel; Nascimento, Janaína de Freitas; Girard, Richard M. B. M.; Silber, Ariel Mariano; Koning, Harry P. de

doi:10.1016/j.bbagen.2018.08.015

Cited by 19 publications

(46 citation statements)

References 74 publications

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“…We have previously shown that it was possible to predict the substrate selectivity of Trypanosoma cruzi ENT transporters by analysing their phylogenetic alignment with ENTs from other kinetoplastids [ 32 ]. Figure 1 A shows a phylogenetic tree of nucleoside and nucleobase transporters from a number of different gene families.…”

Section: Resultsmentioning

confidence: 99%

“…To do so, we used a gain-of-function approach utilizing T. brucei as the expression system, as previously done by us for ENT family transporters of T. congolense , T. cruzi , Leishmania spp. and Trichomonas vaginalis [ 30 , 31 , 32 , 33 ]. We found Tg244440 to have high affinity for oxopurine nucleobases and nucleosides and only low affinity for aminopurines and pyrimidines.…”

Section: Introductionmentioning

confidence: 99%

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A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Campagnaro

Elati

Balaska

et al. 2022

IJMS

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Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Campagnaro

Elati

Balaska

et al. 2022

IJMS

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“…To start assigning gene identities to the observed nucleoside and nucleobase transport activities of T. vaginalis trophozoites, each individual gene was to be expressed in a T. b. brucei strain, TbAT1‐KO, in which we had successfully characterized nucleoside transporters from several other protozoan species including Trypanosoma cruzi (Campagnaro, de Freitas Nascimento, et al., 2018), Trypanosoma congolense (Munday et al., 2013), Leishmania spp (Alzahrani et al., 2017), and Toxoplasma gondii (Campagnaro et al, manuscript in preparation).…”

Section: Resultsmentioning

confidence: 99%

“…We find that the main transporters of T. vaginalis are able to take up purine nucleosides and pyrimidine nucleosides with similar affinity. In keeping with most protozoa no pyrimidine‐specific nucleoside transporters were identified (the sole documented exception is the T. cruzi NT2 transporter (Campagnaro, de Freitas Nascimento, et al., 2018)), although NT‐d did display higher affinity for uridine and thymidine than for adenosine and guanosine. Mixed purine‐pyrimidine nucleoside transporters have been described in Leishmania (Vasudevan et al., 1998) and Toxoplasma gondii (De Koning et al., 2003), whereas purine‐specific nucleoside transporters are the norm in T. brucei (De Koning & Jarvis, 1997, 1999) and Plasmodium falciparum (Quashie et al., 2008).…”

Section: Discussionmentioning

confidence: 97%

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter

Natto

Miyamoto

Munday

et al. 2021

Molecular Microbiology

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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.

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“…Mammal cells use the de novo pathway to obtain purines (16) in their purinosomes (25). In contrast, fungi, bacteria (16), and parasitic protozoa like Plasmodium (26, 27), Entamoeba hystolytica (28, 29), Giardia lamblia (30), Toxoplasma gondii (31, 32), Trichomonas vaginalis (33), Trypanosoma cruzi (34), Trypanosoma brucei (35–37), and Leishmania (38, 39), are purine auxotrophs, which lack the de novo metabolic route and obtain their purines and pyrimidines from imported DNA through salvage pathways.…”

Section: Nucleoside Hydrolases Are Key Enzymes For the Replication Ofmentioning

confidence: 99%

Nucleoside Hydrolase NH 36: A Vital Enzyme for the Leishmania Genus in the Development of T-Cell Epitope Cross-Protective Vaccines

Palatnik-de-Sousa

2019

Front. Immunol.

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NH36 is a vital enzyme of the DNA metabolism and a specific target for anti- Leishmania chemotherapy. We developed second-generation vaccines composed of the FML complex or its main native antigen, the NH36 nucleoside hydrolase of Leishmania (L.) donovani and saponin, and a DNA vaccine containing the NH36 gene. All these vaccines were effective in prophylaxis and treatment of mice and dog visceral leishmaniasis (VL). The FML-saponin vaccine became the first licensed veterinary vaccine against leishmaniasis (Leishmune®) which reduced the incidence of human and canine VL in endemic areas. The NH36, DNA or recombinant protein vaccines induced a Th1 CD4 + IFN-γ + mediated protection in mice. Efficacy against VL was mediated by a CD4 + TNF-α T lymphocyte response against the NH36-F3 domain, while against tegumentary leishmaniasis (TL) a CD8 + T lymphocyte response to F1 was also required. These domains were 36–41 % more protective than NH36, and a recombinant F1F3 chimera was 21% stronger than the domains, promoting a 99.8% reduction of the parasite load. We also identified the most immunogenic NH36 domains and epitopes for PBMC of active human VL, cured or asymptomatic and DTH + patients. Currently, the NH36 subunit recombinant vaccine is turning into a multi-epitope T cell synthetic vaccine against VL and TL.

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Cloning and characterisation of the Equilibrative Nucleoside Transporter family of Trypanosoma cruzi: ultra-high affinity and selectivity to survive in the intracellular niche

Abstract: This study reports the first cloning of T. cruzi purine and pyrimidine transporters, including the first gene encoding a pyrimidine-selective protozoan transporter.

Cited by 19 publications

References 74 publications

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter

Nucleoside Hydrolase NH 36: A Vital Enzyme for the Leishmania Genus in the Development of T-Cell Epitope Cross-Protective Vaccines

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