Molecular Characterization of PGF Synthase-like Protein in Old and New World Species of Leishmania 2α

Abstract

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and spread by the bite of infected sand fly species. Infection of mammals by Leishmania results in increased production of prostaglandins in the host. These prostaglandins which are lipid mediators are responsible for some of the symptoms observed in leishmaniasis including fever and immunosuppression. Prostaglandin F

is among the prostaglandins that are synthesized by Leishmania species. The present study was undertaken to identify and characterize the PGF synthase gene and to

Leishmaniasis is a disease caused by protozoan parasites of the genus Leishmania and spread by the bite of infected sand fly species. Infection of mammals by Leishmania results in increased production of prostaglandins in the host. These prostaglandins which are lipid mediators are responsible for some of the symptoms observed in leishmaniasis including fever and immunosuppression. Prostaglandin F

is among the prostaglandins that are synthesized by Leishmania species. The present study was undertaken to identify and characterize the PGF synthase gene and to identify and characterize the PGF 2α 2α synthase protein in Old and New World Species of Leishmania in order to provide insight into the role of these proteins in the parasite. Isolates of L. major, L. donovani, L. tropica, L. amazonensis, L. braziliensis, L. mexicana and L. chagasi were obtained from the cryobank of American Type Culture Collection (ATCC, Manassas USA) and cultured to obtain promastigotes. Total RNA was extracted from the cell pellets of each of the cultured Leishmania species. To detect gene expression at transcription level, polymerase chain reaction was carried out using L. major PGF synthase gene specific primers and complementary DNA obtained from promastigotes of each of the species mentioned above. To detect expression at translation level, total protein obtained from cultured promastigotes of the above parasites was analyzed on a Western blot using T. bruceispecific rabbit

anti-PGF 2α 2α synthase polyclonal antibodies. At the transcription level, PGF synthase gene expression was detected in Old World species L. major, L. donovani and L. tropica, but was absent in the New World L. amazonensis and L. mexicana. It was expressed at low levels in the New World L. chagasi. Western blot analysis confirmed the presence of PGF 2α synthase - like proteins in Old World and not in New World species. These findings suggest that New World Leishmania may have evolved new ortholog genes to produce PGF 2α . Alternatively, the ancestral PGF 2α 2α synthase gene is present in the New World species but has mutated or been lost due to speciation during evolution. Phenotypic functional studies such as enzyme assays ought to be carried out to ascertain presence and function of PGFS in promastigotes and amastigotes of Old and New World species of Leishmania. 2α above. To detect expression at translation level, total protein obtained from cultured promastigotes of the above parasites was analyzed on a Western blot using T. bruceispecific rabbit

anti-PGF 2α 2α synthase polyclonal antibodies. At the transcription level, PGF synthase gene expression was detected in Old World species L. major, L. donovani and L. tropica, but was absent in the New World L. amazonensis and L. mexicana. It was expressed at low levels in the New World L. chagasi. Western blot analysis confirmed the presence of PGF 2α synthase - like proteins in Old World and not in New World species. These findings suggest that New World Leishmania may have evolved new ortholog genes to produce PGF 2α . Alternatively, the ancestral PGF 2α 2α synthase gene is present in the New World species but has mutated or been lost due to speciation during evolution. Phenotypic functional studies such as enzyme assays ought to be carried out to ascertain presence and function of PGFS in promastigotes and amastigotes of Old and New World species of Leishmania.