Evaluation of Apoptotic Effect of Transgenic Leishmania tropica Expressing MLLO-Bax-Smac Fusion Gene in Infected Macrophages, an In Vitro Study

Maryam  Aghaei; Hossein  Khanahmad; Shahrzad  Aghaei; Akram  Jalali; Zabihollah  Shahmoradi; Nooshin  Hashemi; Akram  Sarmadi; Shima  Aboutalebian; Mohammad Ali  Mohammadi; Sayed Mohsen Hosseini; Seyed Hossein  Hejazi

doi:10.18502/ijpa.v20i3.19611

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 Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, 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

DOI: https://doi.org/10.18502/ijpa.v20i3.19611

Keywords: Leishmaniasis; Transgenic; Apoptosis; Macrophage; Homologous recombi-nation

Abstract

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.