Genetic characterization of glucose transporter function inLeishmania mexicana
| Autor: | Richard J S, Burchmore, Dayana, Rodriguez-Contreras, Kathleen, McBride, Patrick, Merkel, Michael P, Barrett, Govind, Modi, David, Sacks, Scott M, Landfear |
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| Rok vydání: | 2003 |
| Předmět: |
Gene isoform
Time Factors Monosaccharide Transport Proteins Leishmania mexicana Cell Mutant Animals Genetically Modified Mice Acetyltransferases medicine Animals Amastigote Cells Cultured Life Cycle Stages Mice Inbred BALB C Multidisciplinary biology Glucose transporter Biological Sciences biology.organism_classification Subcellular localization Fusion protein Molecular biology Recombinant Proteins Cell biology Kinetics medicine.anatomical_structure Macrophages Peritoneal |
| Zdroj: | Proceedings of the National Academy of Sciences. 100:3901-3906 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.0630165100 |
| Popis: | Both insect and mammalian life cycle stages ofLeishmania mexicanatake up glucose and express all three isoforms encoded by theLmGTglucose transporter gene family. To evaluate glucose transporter function in intact parasites, a null mutant line has been created by targeted disruption of theLmGTlocus that encompasses theLmGT1,LmGT2, andLmGT3genes. This Δlmgtnull mutant exhibited no detectable glucose transport activity. The growth rate of the Δlmgtknockout in the promastigote stage was reduced to a rate comparable with that of WT cells grown in the absence of glucose. Δlmgtcells also exhibited dramatically reduced infectivity to macrophages, demonstrating that expression of LmGT isoforms is essential for viability of amastigotes. Furthermore, WTL. mexicanawere not able to grow as axenic culture form amastigotes if glucose was withdrawn from the medium, implying that glucose is an essential nutrient in this life cycle stage. Expression of eitherLmGT2orLmGT3, but not ofLmGT1, in Δlmgtnull mutants significantly restored growth as promastigotes, but onlyLmGT3expression substantially rescued amastigote growth in macrophages. Subcellular localization of the three isoforms was investigated in Δlmgtcells expressing individual LmGT isoforms. Using anti-LmGT antiserum and GFP-tagged LmGT fusion proteins, LmGT2 and LmGT3 were localized to the cell body, whereas LmGT1 was localized specifically to the flagellum. These results establish that each glucose transporter isoform has distinct biological functions in the parasite. |
| Databáze: | OpenAIRE |
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