<em>In-vitro</em> Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, pathogenic bacteria are initially enclosed within an endosome, from which they break free and escape into the cytosol to avoid lysosomal degradation. Once in the cytosol, bacteria are rapidly tagged with ubiquitin chains by host E3 ligases, marking them for clearance via multiple effector pathways, including autophagy and the proteasome. To delineate the contribution of individual pathways in bacterial elimination, in-vitro reconstitution offers a controlled and unambiguous approach. Here, using Streptococcus pneumoniae (SPN) as a model bacterial pathogen, we present a detailed protocol for its ubiquitination using mammalian cell lysates or pure ubiquitin enzyme complex, followed by treatment with purified VCP/p97-UFD1-NPLOC4 complex to assess its bacteriolytic activity independent of other cellular factors. Overall, this procedure holds the potential to illuminate the function of immunity-linked ubiquitin ligases and effectors in a cell-free context, where dissecting such mechanisms can be challenging in intact cells.