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Identification of coding genes for phospholipids translocation in Leishmania

Grant number: 12/23085-9
Support type:Scholarships in Brazil - Master
Effective date (Start): April 01, 2013
Effective date (End): November 30, 2014
Field of knowledge:Biological Sciences - Parasitology - Protozoology of Parasites
Principal researcher:Lucile Maria Floeter-Winter
Grantee:Carolina de Lima Jorge
Home Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:10/52688-8 - Biochemical, physiological and functional genomics studies of Leishmania-macrophage interaction, AP.TEM

Abstract

Leishmaniasis is a disease caused by protozoa of the genus Leishmania. According to the World Health Organization (WHO) there are around 12 million people infected with this disease in 88 countries in 4 Continents and the disease is considered by the Tropical Diseases Research Program (TDR) one of six most important diseases in Public Health. When inoculated by the insect in the host mammal, the promastigote form presents a strategy that mimicris apoptosis. This strategy results in the transmigration of phosphatidylserine (PS) to the external face of the lipidic bilayer plasmatic membrane. This way the macrophages recognize this apoptotic signal and perform the phagocytosis of the parasites without launching an inflammatory process. The goal of this work is to verify the mechanism responsible for the exposure of PS in Leishmania (L.) amazonensis, by searching in the genome the segment that codifies the molecule responsible for the transmigration of the phospholipids. The experimental strategy used was the transfection of wild parasites with cosmids which constitute the genomic library of the organism. This way, transfected mutants will carry extra copies of Leishmania DNA and under the selection pressure of a marker present in the cosmid, the one carrying the proper information will be able to expose PS in the external membrane in a development phase in which normally PS would not have been exposured. The analysis of the mutants was performed by FACS, since the parasites exposing PS can be marked by annexin-V linked to the FITC. After the fractionation of mutants of interest by Sorter, clones were obtained by platting. The exposure of the phospholipids of each clone was analyzed pointing to clones of interest. Cosmid DNA of those clones will be characterized as a continuation of the project.

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