Ubiquitination and ubiquitination-like modifications are conserved post-translational modifications that influence virtually all physiological processes, including innate immunity. They are controlled by hundreds of ubiquitin (Ub) and Ub-like modifying enzymes that regulate their targets by modulating their stability, localization or activity. In recent years, the importance of these enzymes in C. elegans' innate immunity has been revealed, with the identification of key Ub-related regulators of innate immune pathways, as well as Ub-related effectors in the host response (Garcia-Sanchez et al, 2021). Little is known, however, about the role of these processes in the immune response to the intestinal bacterium Staphylococcus aureus, which is coordinated by the transcription factor TFEB/HLH-30 (Visvikis et al, 2014; Najibi et al, 2016). Our research focuses on deciphering the importance of Ub-modifying enzymes in regulating this immune pathway, as well as their role as effectors in the host response. Firstly, we undertook an RNAi-based screen to identify new immune regulatory enzymes. We generated a fluorescent reporter strain which allowed us to screen the effect of 216 enzymes on the activity of HLH-30 during S. aureus infection. We identified 22 genes whose downregulation reduced reporter fluorescence. To distinguish between specific and more general regulators, we performed a parallel screen using the model of epidermal infection by the fungus Drechmeria coniospora, which triggers an immune response mainly controlled by the
p38 MAPK/PMK-1 pathway (Zugasti et al, 2014; 2016). We found 6 enzymes that are common to both screens; the remaining 16 enzymes are potentially specific regulators of the HLH-30-dependent immune response to S. aureus. Secondly, to identify Ub-related immune effectors important for the response to S. aureus infection, we analyzed previously published RNA-seq data (Visvikis et al, 2014) and determined that 16 out 19 Ub-related enzymes induced during infection are controlled by HLH-30. Focusing on 3 conserved E3 Ub-ligases, we confirmed their HLH-30 dependent induction by qPCR. Interestingly, using survival assays, we could demonstrate their importance in C. elegans' host defense against S. aureus infection suggesting they play a specific effector role in infection. Altogether, these results demonstrate the importance of Ub-related enzymes in HLH-30 dependent host response to S. aureus infection and have revealed specific and general Ub-related mechanisms in the regulation of immune pathways.