Evaluation of transgenic Leishmania major expressing mLLOBAX-EndoG -SMAC in the apoptosis of itself parasite and the infectedmacrophages in vitro and in vivo.

Background and Aim : Cutaneous leishmaniasis is a parasitic infection against which noconfirmed vaccine has been reported so far. Because of escaping the parasite from the host immunesystem or preventing macrophage apoptosis, it seems the expression of pro-apoptotic proteins ofmacrophage (BAX- SMAC) and parasite (EndoG) by transgenic Leishmania major can limitdifferentiation and proliferation of parasites and the ability to cause infection by accelerating theapoptosis process of macrophage and L.major. So, this research investigated the impact oftransgenic L. major including mLLO-BAX- EndoG -SMAC in macrophage and L.major apoptosisaccelerationMethods : The coding sequence mLLO-BAX- EndoG -SMAC was designed and integrated intothe pLexyNeo۲ plasmid. The designed sequence was inserted under the ۱۸srRNA locus into theL. major genome using homologous recombination. Then, mLLO-BAX- EndoG -SMACexpression was studied using the Western blot, and the transgenic parasite pathogenesis wasinvestigated compared to wild-type L. major in vitro and in vivo. Also, the collected macrophage-free supernatants at ۱۲, ۲۴ and ۴۸ h post- stimulation and infected mice sera obtained at ۳, ۴ and۵ week post-infection were subjected to ELISA analysis for TNF-α, IL-۱۰, IL-۱۲ cytokines andBAX, EndoG and Bcl-۲ proteins and compared with ulcer size and parasitic bar of spleen. Thegroups were compared using ANOVA test at the significant level of p < ۰.۰۵.Results : PCR and Western blot results approved the proper integration and expression of themLLO-BAX- EndoG -SMAC under the ۱۸srRNA locus of L. major, respectively. The flowcytometry results revealed faster apoptosis of transgenic Leishmania-infected macrophagescompared to wild-type parasite-infected macrophages. Also, in vitro, at ۱۲h, apoptosis promotionof macrophage and transgenic L.major was correlated with increased production of IL-۱۲, TNF-?,BAX, Endo G and reduced production of IL-۱۰ and Bcl-۲. In vivo, increased and decreased levelsof the mentioned cytokines were associated to the reduction of the lesion size and the parasiticburden of spleen in transgenic L.major- infected mice compared to wild type- infected mice.Conclusion : This study recommended transgenic L. major including mLLO-BAX- EndoG -SMAC construct as a pilot model for providing a protective vaccine against leishmaniasis.

کلیدواژه ها:

Leishmania major ، transgenic ، apoptosis ، macrophage