Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 20 3 2025 09 01 366 377 Maryam Aghaei Skin Diseases and Leishmaniasis Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran Hossein Khanahmad Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Shahrzad Aghaei 1. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 2. Department of Molecular Medicine, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran Akram Jalali Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran Zabihollah Shahmoradi Skin Diseases and Leishmaniasis Research Centre, Department of Dermatology, Isfahan University of Medical Sciences, Isfahan, Iran Nooshin Hashemi School of Medicine, Bam University of Medical Sciences, Bam, Iran Akram Sarmadi Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Shima Aboutalebian Department of Medical Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Mohammad Ali Mohammadi Research Center for Hydatid Disease in Iran, Kerman University of Medical Sciences, Kerman, Iran Sayed Mohsen Hosseini Department of Biostatistics & Epidemiology, School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran Seyed Hossein Hejazi Skin Diseases and Leishmaniasis Research Center, Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran 2023 04 04 2025 01 12 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. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3917 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3917/1443