THE FUNCTIONAL ROLE OF DROSOPHILA MELANOGASTER TEN ELEVEN TRANSLOCATION PROTEIN dTET ON HUMORAL IMMUNITY

Abstract

Introduction: DNA demethylation, an epigenetic mechanism mediated by Tet proteins, is commonly known to modulate gene expression. Tet proteins in mammals are involved in the development and differentiation of various cells, including adaptive immune cells. In Drosophila melanogaster, dTet the unique mammalian Tet homolog is known for its vital role in development and embryogenesis as dTet-deficient flies die early during embryogenesis. While we have previously shown that dTet is expressed in the fat body, its role in fly immunity is not well understood. Therefore, this study is the first to focus on assessing the functional impact of dTet on innate immunity and more specifically the humoral immune pathways regulated by Imd and Toll pathways. Methods: dTet in Drosophila fat body cells, was knocked down using a fat body-specific driver (C564-GAL4) and RNAi-mediated gene knockdown. Loss of dTet expression was validated by RT-qPCR. Furthermore, relative expression levels of diptericin and drosomycin, the major antimicrobial peptides (AMPs) released upon the activation of Imd and Toll humoral immune pathways, respectively were measured in dTet-deficient flies. Survival assays were performed in dTet-RNAi flies before and after Gram-negative (Escherichia coli) and Gram-positive (Enterococcus faecalis) bacterial challenge to assess the impact of dTet knockdown on the activity of Imd and Toll pathways against the bacterial challenge. Results: dTet mRNA levels were significantly downregulated upon dTet knockdown in fat body cells. Upon knocking down dTet, the relative expression of AMPs (diptericin and drosomycin) was significantly upregulated. Moreover, although the E.coli challenge in dTet-deficient flies severely affected fly survival; challenging dTet-deficient flies with E. faecalis had an unclear survival pattern that requires further investigations. Conclusions: In this study, we have characterized the impact of dTet knockdown on fly humoral immune pathways (Imd and Toll) and identified that dTet knockdown severely affected fly survival upon Gram-negative bacterial challenge pointing towards a role for dTet in regulating Imd humoral immune responses. Future studies will be required to elucidate whether dTet has a role in Toll-mediated humoral responses and understand the mechanisms through which this is achieved.