Evaluation of Apoptotic Effect of Transgenic Leishmania tropica Expressing MLLO-Bax-Smac Fusion Gene in Infected Macrophages, an In Vitro Study
Maryam Aghaei1, Hossein Khanahmad2, Shahrzad Aghaei2,3
1Skin Diseases and Leishmaniasis Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran.
Background:
In the leishmaniasis parasitic infection, parasite escapes from the immune system of host or prevents macrophage apoptosis. It seems generating transgenic parasites to express proapoptotic proteins can accelerate the apoptosis of infected macrophage and prevent Leishmania differentiation. Hence, we investigated the efficacy of transgenic L. tropica expressing mLLO-BAX-SMAC in expediting macrophage apoptosis.
Methods:
This study was performed at the Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences (2022 to 2023), Isfahan, Iran. mLLO-Bax-Smac coding sequence cloned in the pLexyNeo2 was entered into the L. tropica genome using homologous recombination. PCR, Western blot, and hemolysis tests were used to confirm integration accuracy. In addition, the apoptosis percentage of transgenic L. tropica- infected macrophages was assayed by flow-cytometry.
Results:
The proper integration of the mLLO-Bax-Smac fragment into the 18srRNA locus of L. tropica and mLLO -BAX-SMAC fusion protein expression was approved. Furthermore, results of flow cytometry showed the mean percentage of apoptosis among the groups is different and transgenic L. tropica leads to a decrease in the apoptosis time of infected macrophages compared to the wild type of Leishmania and to an increase in the immune system response.
Conclusion:
The data suggest the transgenic L. tropica can be used as an experimental model to expedite apoptosis of Leishmania-infected macrophage.
