SUMOylation regulates the function of Drosophila Jun and helps maintain gut immune homeostasis

Abstract

To invoke a robust immune response, the host must recognise the invading pathogen and produce a battery of nullifying agents to neutralize the infection. One key feature for a successful host-defence is to find the right balance between positive and negative regulation of several immune effector pathways. There is ample evidence to suggest that post-translational modifications (PTMs) play a crucial role in the regulation of immune regulatory pathways during infection. Small Ubiquitin-like Modifier (SUMO) is one such post-translational modifier that has been implicated as an important regulator of the immune response, in recent years. To understand the role of SUMOylation in the regulation of innate immunity in Drosophila, as a first step, we identified, using quantitative mass spectrometry, a set of 700 proteins that display significantly altered SUMOylation status upon infectioninS2 cells. These SUMO targets include several important transcriptional regulators, not the least of which are the Fos/Jun heterodimer, also known as the AP1 complex. Here, we validate the SUMO conjugation of the Drosophila AP1 complex and attempt to uncover its role in host defence. Both Jun (Jra; Jun related antigen) and Fos (Kay; Kayak) are SUMO conjugated as tested using in-vitro and in-vivo SUMO conjugation assays. SUMO conjugation resistant (SCR) variants of both Fos and Jun were generated by uncovering SUMO acceptor lysine residues and replacing these with arginine residues. Further, we have used CRISPR/Cas9 technology to edit the Drosophila genome and have generated a JraSCR mutant fly line. Using specific knockdowns, I established Jra as a negative regulator of the gut immune response in Drosophila, a role that is previously known. Flies expressing JraSCR are sensitive to gut infection by gram-negative bacteria, P. entomophila, and succumb early. 3’mRNA sequencing analysis of the guts of JraSCR flies revealed that a subset of defence genes are insufficiently activated post-infection. The genes affected include transcriptional targets of Jra like Chinmo, Fkh, Ets21C, and also the NFkB factors Relish and Dorsal. Our data suggests that Jra is a negative regulator of the gut immune response and SUMO conjugation of Jra attenuates the negative regulation by Jra to evoke a strong immune response. We propose that the SUMOylation of Jra is a key regulatory step that fine-tunes the activation of innate immune pathways during infection and maintains gut immune homeostasis