Maryam Aghaei*, Hossein Khanahmad, Shahrzad Aghaei, Sayed Mohsen Hosseini, Mahin Farahmand, and Seyed Hossein Hejazi
Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran
Received: July 20, 2021 Accepted: July 22, 2021 Published: July 22, 2021
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 BAXSMAC can be used as an experimental model for developing vaccination against leishmaniasis.
Keywords: Apoptosis, Electroporation, Homologous recombination, Leishmaniasis, Transgenic