Interferon-Stimulated Gene 15 (ISG15) is a ubiquitin-like modification involved in host defense against viral, bacterial and fungal infections. Cells lacking ISG15 are strongly susceptible to viral and intracellular bacterial pathogens indicating ISG15 as a key factor in cell-autonomous immunity. Similar to ubiquitin, ISG15 covalently conjugates to pathogen and host proteins, however, the molecular impact of ISG15 on its substrate proteins remains elusive. During viral infection, ISG15 conjugates to newly translated proteins in a co-translational manner, thus targeting viral proteins and impeding their function in a loss-of-function model. However, this model does not explain ISG15’s antibacterial activity and recent discoveries suggest that ISG15 conjugation to host proteins might also lead to gain-of-function effects, activating host effector proteins by oligomerization or by changing their subcellular localization to target for instance invading bacterial pathogens. The present proposal aims to investigate this hypothesis by combining advanced molecular, biochemical and imaging tools on cells infected by the intracellular bacterial model pathogen Listeria monocytogenes to gain fundamental insight into the ISGylation pathway and reveal novel antibacterial host proteins.