Somtochukwu S. Onwah
Conference 2022 Live Talk
The role of dihydrolipoyl dehydrogenase (DLD) in the immunopathogenesis of Leishmania major
Authors and Affiliations
Somtochukwu Stella Onwah1, Zhirong Mou1, Ping Jia1 Jude Uzonna1
1.Department of Immunology, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB.
Cutaneous leishmaniasis (CL) caused by numerous Leishmania species including Leishmania major, leads to a range of diseases, from self-healing lesions to chronic disfiguring disease. Protection is achieved from IFN-g producing CD4+ T cell activation on Leishmania infected macrophages. Dihydrolipoyl dehydrogenase (DLD) is a critical mitochondrial enzyme in eukaryotic cells including Leishmania known to modulate metabolic activities. In different pathogenic organisms, DLD is a promising therapeutic target. The role and contribution of DLD in L. major immunopathogenesis is currently not known. We hypothesize that DLD is a virulence factor and its deficiency in L. major will result in an attenuated disease pathology and altered host immune response.
To generate DLD deficient L. major, an all-in-one plasmid (pLDCN) containing two short oligonucleotide sequences (guide RNA) complementary to the DLD gene in L. major was introduced such that upon expression, Cas9 initiates cleavage. A homology-directed repair mechanism allowed for the introduction of a donor DNA (bleomycin) PCR product into the deleted site. I functionally validated DLD gene deletion in axenic culture, bone marrow-derived macrophages and in mice.
Deficiency of DLD in L. major was confirmed by PCR and in vivo by the absence of DLD-specific CD4+ T cells from splenocytes of mice infected with DLD deficient parasites using DLD-specific tetramers. Growth kinetics in axenic culture and macrophages show that deficiency of DLD gene products results in reduced proliferation in comparison to wild-type (WT) parasites. Mice infected with DLD deficient parasites in the footpad had no observable lesion and significantly reduced parasite burden compared to WT-infected animals. The frequency of cytokine (IFN-g and TNF)-producing CD4+ T cells in spleens of mice infected with DLD deficient parasites was significantly lower than those from their WT counterparts. Cells from mice infected with DLD deficient parasites produced significantly reduced levels of these cytokines in the culture supernatant following in vitro restimulation with soluble Leishmania Ag.
These findings suggests that DLD in L. major is a critical metabolic enzyme for intracellular survival both in axenic culture and inside macrophages. Since DLD deficient parasites did not induce pathologies in mice but alters host immune response indicates that they could be good vaccine candidates