Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

Campagnaro, Gustavo Daniel; Alzahrani, Khalid J.; Munday, Jane C.; Koning, Harry P. de

doi:10.1016/j.molbiopara.2018.01.005

Cited by 16 publications

(17 citation statements)

References 63 publications

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“…The transport of purines was measured using tritiated guanine (20 Ci/mmol; American Radiolabeled Chemicals) and hypoxanthine (12.8 Ci/mmol; Perkin-Elmer) diluted in assay buffer (AB; 33 mM HEPES, 98 mM NaCl, 4.6 mM KCl, 0.5 mM CaCl 2 , 0.07 mM MgSO 4 , 5.8 mM NaH 2 PO 4 , 0.03 mM MgCl 2 , 23 mM NaHCO 3 , 14 mM D-glucose, pH 7.3), in the presence or absence of potential transporter inhibitors at a predetermined concentration, as described previously for Trypanosoma brucei and Leishmania [ 65 , 66 ].…”

Section: Methodsmentioning

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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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: Methodsmentioning

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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“…Transport assays with T. vaginalis trophozoites and T. brucei bloodstream forms were performed identically and essentially as described previously for T. brucei (Wallace et al., 2002) and Leishmania promastigotes (Alzahrani et al., 2017). Briefly, cells of either species were grown in larger culture flasks (75 cm 2 U‐shaped Canted Neck Culture Flask with Vent Cap (Corning) for T. brucei , 25 ml glass Universals, completely filled with medium for T. vaginalis ), harvested and washed twice by centrifugation (10 min, 1,000 × g , room temperature) into an assay buffer (AB (Campagnaro, Alzahrani, et al., 2018)) and resuspended at 10 8 cells/mL. Aliquots of 100 µl (i.e.…”

Section: Methodsmentioning

confidence: 99%

“…A single report from 1988 describes two nucleoside transport activities, one that transports all nucleosides and one selective for adenosine, guanosine and uridine (Harris et al., 1988); neither was inhibited by nucleobases, although adenine and guanine have both been shown to be incorporated into the T. vaginalis nucleotide pool (Munagala & Wang, 2003). Yet, the genome of T. vaginalis contains nine genes of the Equilibrative Nucleoside Transporter (ENT) family (TrichDB.org), to which, to date, all protozoan nucleoside and nucleobase transporters have been attributed (Campagnaro & De Koning, 2020; De Koning et al., 2005), although there are some indications that there may be some protozoan nucleobase transport activities that are not encoded by ENT genes (Campagnaro, Alzahrani, et al., 2018; De Koning, 2007). We therefore performed a comprehensive examination of nucleoside and nucleobase transport in T. vaginalis trophozoites with the objective to begin the process of assigning specific transport activities to individual genes, as well assessing their relative levels of expression and their regulation in the presence and absence of substrate and feeder cells.…”

Section: Introductionmentioning

confidence: 99%

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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“…is a purine auxotroph [32,33], being incapable of synthesizing purine nucleotides de novo, and relying on uptake from the hosts to afford its metabolic needs via the purine salvage pathway. Trypanosomes incorporate purine nucleobases and purine analogs by a highly selective nucleobase/proton symporter system with differential expression during procyclic and bloodstream stages [34][35][36][37]. Once inside the cell, the purine nucleobases and nucleosides are further converted into nucleotides by enzymes of the salvage pathway, including three purine phosphoribosyltransferases which have some overlapping substrate specificity.…”

Section: Introductionmentioning

confidence: 99%

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active

2022

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Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based prodrug activation.

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Trypanosoma brucei bloodstream forms express highly specific and separate transporters for adenine and hypoxanthine; evidence for a new protozoan purine transporter family?

Cited by 16 publications

References 63 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

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active

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