Effect of transgenic Leishmania major expressing mLLO-Bax-Smac fusion gene in the apoptosis of the infected macrophages

Document Type : Original Article

Authors

1 Skin Diseases and Leishmaniasis Research Centre, 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 Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

4 Department of Molecular Medicine, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran

5 Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

6 Department of Biostatistics & Epidemiology, School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran

7 Skin Diseases and Leishmaniasis Research Center, Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract

Objective(s): Leishmaniasis is a complex infection against which no confirmed vaccine has been reported so far. Transgenic expression of proteins involved in macrophage apoptosis-like BAX through the parasite itself accelerates infected macrophage apoptosis and prevents Leishmania differentiation. So, in the present research, the impact of the transgenic Leishmania major including mLLO-BAX-SMAC proapoptotic proteins was assayed in macrophage apoptosis acceleration. 
Materials and Methods: The coding sequence mLLO-Bax-Smac was designed and integrated into the pLexyNeo2 plasmid. The designed sequence was inserted under the 18srRNA locus into the L. major genome using homologous recombination. Then, mLLO-BAX-SMAC expression was studied using the Western blot, and the transgenic parasite pathogenesis was investigated compared with wild-type L. major in vitro and also in vivo. 
Results: Western blot and PCR results approved mLLO-BAX-SMAC expression and proper integration of the mLLO-Bax-Smac fragment under the 18srRNA locus of L. major, respectively. The flow cytometry results revealed faster apoptosis of transgenic Leishmania-infected macrophages compared with wild-type parasite-infected macrophages. Also, the mild lesion with the less parasitic burden of the spleen was observed only in transgenic Leishmania-infected mice. The delayed progression of leishmaniasis was obtained in transgenic strain-injected mice after challenging with wild-type Leishmania. 
Conclusion: This study recommended transgenic L. major including mLLO-BAX-SMAC construct as a pilot model for providing a protective vaccine against leishmaniasis. 

Keywords

References

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Iranian Journal of Basic Medical Sciences
Volume 24, Issue 12
December 2021
Pages 1666-1675

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