Maryam Aghaei1, Hossein Khanahmad2, Shahrzad Aghaei3, Sayed Mohsen Hosseini4, Mahin Farahmand5, Seyed Hossein Hejazi1,6. 1. Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. 2. Department of Genetics and molecular biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran. 3. Department of Molecular Medicine, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran. 4. Department of Biostatistics & Epidemiology, School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran. 5. Department of Parasitology, Pasteur Institute of Iran, Tehran, Iran. 6. Skin Diseases and Leishmaniasis Research Center, Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.
Abstract
BACKGROUND: Leishmaniasis is an important infectious disease that develops because of escaping parasite from the host immune system or preventing host macrophages apoptosis. Recently, the development of transgenic methods and the manipulation of the parasite genome has provided many advantages. So, in this study, the effect of the transgenic Leishmania infantum expressing mLLO-BAX-SMAC proteins was examined in accelerating host cell apoptosis. METHOD: The entire coding sequence of designed codon-optimized mLLO-Bax-Smac was cloned in the pLexyNeo2 vector and integrated downstream of the 18srRNA locus of L infantum genome by homologous recombination. Next, the expression of mLLO-BAX-SMAC fusion protein was evaluated by the Western blotting technique and the pathogenesis of transgenic parasite was surveyed in vitro and in vivo. RESULTS: The results of PCR and Western blot confirmed proper integration and expression of mLLO-Bax-Smac sequence into the 18srRNA locus of L infantum. Flow cytometry showed accelerating apoptosis of transgenic Leishmania-infected macrophages compared to wild-type parasite. Also, transgenic parasites were less virulent as a fewer parasitic burden was found in the spleen and liver of transgenic-infected mice compared to the control. CONCLUSION: The data suggested that the transgenic L infantum expressing BAX-SMAC can be used as an experimental model for developing vaccination against leishmaniasis.
BACKGROUND:Leishmaniasis is an important infectious disease that develops because of escaping parasite from the host immune system or preventing host macrophages apoptosis. Recently, the development of transgenic methods and the manipulation of the parasite genome has provided many advantages. So, in this study, the effect of the transgenic Leishmania infantum expressing mLLO-BAX-SMAC proteins was examined in accelerating host cell apoptosis. METHOD: The entire coding sequence of designed codon-optimized mLLO-Bax-Smac was cloned in the pLexyNeo2 vector and integrated downstream of the 18srRNA locus of L infantum genome by homologous recombination. Next, the expression of mLLO-BAX-SMAC fusion protein was evaluated by the Western blotting technique and the pathogenesis of transgenic parasite was surveyed in vitro and in vivo. RESULTS: The results of PCR and Western blot confirmed proper integration and expression of mLLO-Bax-Smac sequence into the 18srRNA locus of L infantum. Flow cytometry showed accelerating apoptosis of transgenic Leishmania-infected macrophages compared to wild-type parasite. Also, transgenic parasites were less virulent as a fewer parasitic burden was found in the spleen and liver of transgenic-infectedmice compared to the control. CONCLUSION: The data suggested that the transgenic L infantum expressing BAX-SMAC can be used as an experimental model for developing vaccination against leishmaniasis.