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Identification of Leishmania mexicana amastigote-specific genes and proteins Bellatin, Jaime Antonio


The leishmaniases are parasitic human diseases that constitute a public health problem in many parts of the world. The diseases are caused by the protozoan Leishmania that as amastigotes are intracellular parasites of the host's macrophages. The efforts to control these diseases will be greatly aided by a better understanding of the Leishmania amastigote and its complex relationship with its mammalian host. Leishmania genes that are preferentially expressed in amastigotes encode proteins that are likely involved in amastigote-specific functions. The first approach used in this thesis was the identification and isolation of amastigote surface proteins. The surface proteins of the amastigote are likely to have a significant role in the host-parasite relationship as they stand in the interphase between the two organisms. This approach was pursued by the generation of monoclonal antibodies directed against the surface of amastigotes. Major surface proteins of L. mexicana axenicculture amastigotes were found to be glycoprotein 63 and a novel protein complex, consisting of three polypeptides of 110, 86 and 70 kDa. This protein complex appeared to be amastigote-specific as it was not detected on the surface of L. mexicana promastigotes and appeared on the surface of cultured Leishmania when they differentiated from promastigotes to amastigotes. The second approach of the thesis was the identification and characterization of amastigote-specific genes by subtractive hybridization. Two amastigote-specific genes were identified and sequenced: A600 and A850. A600 was abundantly expressed in the amastigotes and found to code for a novel small polypeptide of 93 amino acids, the first 42 of which were a predicted signal peptide, implying that the A600 polypeptide may be secreted by the amastigotes. The other amastigote-specific gene identified, A850, encoded a β- tubulin isogene. The A850 mRNA had a unique 3' UTR that hybridized with two copies out of the multiple p-tubulin genes. The amino acid sequence of the A850 gene was compared to that of the three other reported Leishmania p-tubulin genes and the four genes were found to be highly conserved with variable amino acids at only a few defined positions.

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