Ayra Diandra Carvalho-de-Araújo, Luiz Fernando Carvalho-Kelly, Claudia F. Dick, José Roberto Meyer-Fernandes
{"title":"十二指肠贾第虫体内无机磷酸盐转运蛋白及其在ATP合成中的可能作用","authors":"Ayra Diandra Carvalho-de-Araújo, Luiz Fernando Carvalho-Kelly, Claudia F. Dick, José Roberto Meyer-Fernandes","doi":"10.1016/j.molbiopara.2022.111504","DOIUrl":null,"url":null,"abstract":"<div><p><span><em>Giardia duodenalis</em></span><span><span> is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as </span>giardiasis. Similar to other parasites, </span><em>G. duodenalis</em> must take advantage of environmental resources to survive, such as inorganic phosphate (P<sub>i</sub>) availability. P<sub>i</sub><span> is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis<span> of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H</span></span><sup>+</sup>:P<sub>i</sub>-type ORF sequence in the <em>G. duodenalis</em> genome (GenBank ID: GL50803_5164), named <em>GdPho84,</em> which is homologous to <span><em>Saccharomyces cerevisiae</em></span> PHO84. In trophozoites, P<sub>i</sub> transport was linear for up to 15 min, and the cell density was 3 × 10<sup>7</sup> cells/ml. Physiological variations in pH (6.4–8.0) did not influence P<sub>i</sub> uptake. This P<sub>i</sub> transporter had a high affinity, with K<sub>0.5</sub> = 67.7 ± 7.1 µM P<sub>i</sub>. SCH28080 (inhibitor of H<sup>+</sup>, K<sup>+</sup><span>-ATPase), bafilomycin A</span><sub>1</sub> (inhibitor of vacuolar H<sup>+</sup>-ATPase), and FCCP (H<sup>+</sup> ionophore) were able to inhibit P<sub>i</sub> transport, indicating that an H<sup>+</sup> gradient in the cell powered uphill P<sub>i</sub> movement. PAA, an H<sup>+</sup>-dependent P<sub>i</sub><span> transport inhibitor, reduced cell proliferation, P</span><sub>i</sub> transport activity, and GdPHO48 mRNA levels. P<sub>i</sub> starvation stimulated membrane potential-sensitive P<sub>i</sub> uptake coupled to H<sup>+</sup> fluxes, increased <em>GdPho84</em> expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize P<sub>i</sub> as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized P<sub>i</sub><span> can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits P</span><sub>i</sub> influx. Together, these results reinforce the hypothesis that P<sub>i</sub> is a crucial nutrient for <em>G. duodenalis</em> energy metabolism.</p></div>","PeriodicalId":18721,"journal":{"name":"Molecular and biochemical parasitology","volume":null,"pages":null},"PeriodicalIF":1.4000,"publicationDate":"2022-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Inorganic phosphate transporter in Giardia duodenalis and its possible role in ATP synthesis\",\"authors\":\"Ayra Diandra Carvalho-de-Araújo, Luiz Fernando Carvalho-Kelly, Claudia F. Dick, José Roberto Meyer-Fernandes\",\"doi\":\"10.1016/j.molbiopara.2022.111504\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p><span><em>Giardia duodenalis</em></span><span><span> is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as </span>giardiasis. Similar to other parasites, </span><em>G. duodenalis</em> must take advantage of environmental resources to survive, such as inorganic phosphate (P<sub>i</sub>) availability. P<sub>i</sub><span> is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis<span> of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H</span></span><sup>+</sup>:P<sub>i</sub>-type ORF sequence in the <em>G. duodenalis</em> genome (GenBank ID: GL50803_5164), named <em>GdPho84,</em> which is homologous to <span><em>Saccharomyces cerevisiae</em></span> PHO84. In trophozoites, P<sub>i</sub> transport was linear for up to 15 min, and the cell density was 3 × 10<sup>7</sup> cells/ml. Physiological variations in pH (6.4–8.0) did not influence P<sub>i</sub> uptake. This P<sub>i</sub> transporter had a high affinity, with K<sub>0.5</sub> = 67.7 ± 7.1 µM P<sub>i</sub>. SCH28080 (inhibitor of H<sup>+</sup>, K<sup>+</sup><span>-ATPase), bafilomycin A</span><sub>1</sub> (inhibitor of vacuolar H<sup>+</sup>-ATPase), and FCCP (H<sup>+</sup> ionophore) were able to inhibit P<sub>i</sub> transport, indicating that an H<sup>+</sup> gradient in the cell powered uphill P<sub>i</sub> movement. PAA, an H<sup>+</sup>-dependent P<sub>i</sub><span> transport inhibitor, reduced cell proliferation, P</span><sub>i</sub> transport activity, and GdPHO48 mRNA levels. P<sub>i</sub> starvation stimulated membrane potential-sensitive P<sub>i</sub> uptake coupled to H<sup>+</sup> fluxes, increased <em>GdPho84</em> expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize P<sub>i</sub> as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized P<sub>i</sub><span> can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits P</span><sub>i</sub> influx. Together, these results reinforce the hypothesis that P<sub>i</sub> is a crucial nutrient for <em>G. duodenalis</em> energy metabolism.</p></div>\",\"PeriodicalId\":18721,\"journal\":{\"name\":\"Molecular and biochemical parasitology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.4000,\"publicationDate\":\"2022-09-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Molecular and biochemical parasitology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0166685122000585\",\"RegionNum\":4,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and biochemical parasitology","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0166685122000585","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Inorganic phosphate transporter in Giardia duodenalis and its possible role in ATP synthesis
Giardia duodenalis is a flagellated protozoan that inhabits vertebrate host intestines, causing the disease known as giardiasis. Similar to other parasites, G. duodenalis must take advantage of environmental resources to survive, such as inorganic phosphate (Pi) availability. Pi is an anionic molecule and an essential nutrient for all organisms because it participates in the biosynthesis of biomolecules, energy storage, and cellular structure formation. The first step in Pi metabolism is its uptake through specific transporters on the plasma membrane. We identified a symporter H+:Pi-type ORF sequence in the G. duodenalis genome (GenBank ID: GL50803_5164), named GdPho84, which is homologous to Saccharomyces cerevisiae PHO84. In trophozoites, Pi transport was linear for up to 15 min, and the cell density was 3 × 107 cells/ml. Physiological variations in pH (6.4–8.0) did not influence Pi uptake. This Pi transporter had a high affinity, with K0.5 = 67.7 ± 7.1 µM Pi. SCH28080 (inhibitor of H+, K+-ATPase), bafilomycin A1 (inhibitor of vacuolar H+-ATPase), and FCCP (H+ ionophore) were able to inhibit Pi transport, indicating that an H+ gradient in the cell powered uphill Pi movement. PAA, an H+-dependent Pi transport inhibitor, reduced cell proliferation, Pi transport activity, and GdPHO48 mRNA levels. Pi starvation stimulated membrane potential-sensitive Pi uptake coupled to H+ fluxes, increased GdPho84 expression, and reduced intracellular ATP levels. These events indicate that these cells had an increased capacity to internalize Pi as a compensatory mechanism compared to cells maintained in control medium conditions. Internalized Pi can be used in glycolytic metabolism once iodoacetamide (GAPDH inhibitor) inhibits Pi influx. Together, these results reinforce the hypothesis that Pi is a crucial nutrient for G. duodenalis energy metabolism.
期刊介绍:
The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths and their interactions with both the definitive and intermediate host. The main subject areas covered are:
• the structure, biosynthesis, degradation, properties and function of DNA, RNA, proteins, lipids, carbohydrates and small molecular-weight substances
• intermediary metabolism and bioenergetics
• drug target characterization and the mode of action of antiparasitic drugs
• molecular and biochemical aspects of membrane structure and function
• host-parasite relationships that focus on the parasite, particularly as related to specific parasite molecules.
• analysis of genes and genome structure, function and expression
• analysis of variation in parasite populations relevant to genetic exchange, pathogenesis, drug and vaccine target characterization, and drug resistance.
• parasite protein trafficking, organelle biogenesis, and cellular structure especially with reference to the roles of specific molecules
• parasite programmed cell death, development, and cell division at the molecular level.