Leishmania are parasitic protozoa that cause a major, life-threatening disease which afflicts millions of people living in tropical regions of the globe. The major objective of this proposal is to study several genes in Leishmania parasites whose expression is restricted to the stage of the life cycle that inhabits the insect vector. These genes code for membrane transport proteins which are apparently involved in the parasite's adaption to the environment of the insect gut, probably by allowing the parasite to utilize nutrients which are available in the insect but not in the mammalian host. One of these genes, designated Pro-1, codes for a protein related in sequence known glucose transport proteins from mammals, yeast and bacteria and which probably transports either glucose or another sugar. The ligand that is transported by the Pro-1 protein will be determined by transfecting Leishmania parasites with this cloned gene under conditions which will allow accumulation of many copies of the transformed gene. These transfected parasites will then be assayed for increased ability to transport various radiolabeled sugar ligands, compared to untransfected parasites. In addition, the location of the Pro-1 protein within the parasite will be investigated by immunoelectron microscopy to determine whether it is located in the plasma membrane and whether it is uniformly distributed or localized to a specific region of the membrane. Finally, the transcription of the Pro-1 gene will be investigated to determine where its promoter is located. Understanding transcription of the Pro-1 gene may help reveal how this gene is regulated during the parasite life cycle, being expressed in the insect from parasites but not in the form that infects the mammalian host. Two other genes that code for related membrane transport proteins, designated D1 and D2, will also be studied in detail. Complete genomic copies of D1 and D2 will be isolated and sequenced to determine the deduced amino acid sequence of each protein. The ligands which are transported by D1 and D2 will be determined, either by transfecting parasites with many copies of each gene and assaying for increased transport activity or be disrupting each gene and assaying for loss of specific transport function. Together these studies will advance our understanding of how genes are regulated during the parasite life cycle, allowing the parasite to adapt both its host and insect vector, and of how membrane transport proteins function to allow the parasite to survive in its changing environment.
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