title: Trichomonas vaginalis NTPDase and ecto-5'-nucleotidase hydrolyze guanine nucleotides and increase extracellular guanosine levels under serum restriction.
abstract: Trichomonas vaginalis is the aethiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease in the world. The purinergic signaling pathway is mediated by extracellular nucleotides and nucleosides that are involved in many biological effects as neurotransmission, immunomodulation and inflammation. Extracellular nucleotides can be hydrolyzed by a family of enzymes known as ectonucleotidases including the ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) family which hydrolyses nucleosides triphosphate and diphosphate as preferential substrates and ecto-5'-nucleotidase which catalyzes the conversion of monophosphates into nucleosides. In T. vaginalis the E-NTPDase and ecto-5'-nucleotidase activities upon adenine nucleotides have already been characterized in intact trophozoites but little is known concerning guanine nucleotides and nucleoside. These enzymes may exert a crucial role on nucleoside generation, providing the purine sources for the synthesis de novo of these essential nutrients, sustaining parasite growth and survival. In this study, we investigated the hydrolysis profile of guanine-related nucleotides and nucleoside in intact trophozoites from long-term-grown and fresh clinical isolates of T. vaginalis. Knowing that guanine nucleotides are also substrates for T. vaginalis ectoenzymes, we evaluated the profile of nucleotides consumption and guanosine uptake in trophozoites submitted to a serum limitation condition. Results show that guanine nucleotides (GTP, GDP, GMP) were substrates for T. vaginalis ectonucleotidases, with expected kinetic parameters for this enzyme family. Different T. vaginalis isolates (two from the ATCC and nine fresh clinical isolates) presented a heterogeneous hydrolysis profile. The serum culture condition increased E-NTPDase and ecto-5'-nucleotidase activities with high consumption of extracellular GTP generating enhanced GDP, GMP and guanosine levels as demonstrated by HPLC, with final accumulation of the nucleoside. The transcript levels of the five TvNTPDases gene sequences were analyzed by qRT-PCR and the highest gene expressions were found for TvNTPDase 2 and 4. The extracellular guanosine uptake was observed as (13C)GTP nucleotide into parasite DNA and it was lower than that observed for adenosine, labeled as (13C)ATP. These findings indicate the T. vaginalis preference for adenosine uptake and the accumulation of guanosine in the extracellular milieu, corroborating with HPLC data. Our data demonstrate, for the first time, the cascade of guanine nucleotides in T. vaginalis and open possibilities on the study of guanine-related purines other than the classical intracellular activity of G proteins for signal transduction.
authors.affiliation: Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil.
authors.forename: Camila Braz
authors.initials: CB
authors.lastname: Menezes
authors.affiliation: Laboratório de Toxicologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil.
authors.forename: Juliano
authors.initials: J
authors.lastname: Durgante
authors.affiliation: Instituto do Petróleo e dos Recursos Naturais, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil.
authors.forename: Rafael Rodrigues
authors.initials: RR
authors.lastname: de Oliveira
authors.affiliation: Instituto do Petróleo e dos Recursos Naturais, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil.
authors.forename: Victor Hugo Jacks Mendes
authors.initials: VH
authors.lastname: Dos Santos
authors.affiliation: Instituto do Petróleo e dos Recursos Naturais, Pontifícia Universidade Católica do Rio Grande do Sul, Av. Ipiranga 6681, 90619-900 Porto Alegre, RS, Brazil.
authors.forename: Luiz Frederico
authors.initials: LF
authors.lastname: Rodrigues
authors.affiliation: Laboratório de Toxicologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil.
authors.forename: Solange Cristina
authors.initials: SC
authors.lastname: Garcia
authors.affiliation: Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil.
authors.forename: Odelta
authors.initials: O
authors.lastname: Dos Santos
authors.affiliation: Laboratório de Pesquisa em Parasitologia, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Av. Ipiranga 2752, 90610-000 Porto Alegre, RS, Brazil. Electronic address: tiana.tasca@ufrgs.br.
authors.forename: Tiana
authors.initials: T
authors.lastname: Tasca
mesh_terms: D015720:5'-Nucleotidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D015870:Gene Expression; D006150:Guanine Nucleotides / Q000378:metabolism*; D006151:Guanosine / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D014246:Trichomonas vaginalis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; D006151:Guanosine; D006160:Guanosine Triphosphate; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases
keywords: Ecto-5′-nucleotidase; Ectonucleoside triphosphate diphosphohydrolase; Guanosine; Serum limitation; Trichomonas vaginalis