A functional screen for bacterial ubiquitin regulation identifies an unusual Pseudomonas aeruginosa E3 ligase

Bacterial pathogens have adapted secreted effector proteins that hijack host ubiquitination to gain control over host responses. These effectors can exhibit, for example, E3 ligase or deubiquitinase activities, often without any homology to eukaryotic regulators. Such convergence in function poses a challenge to the discovery of additional ubiquitin-targeted virulence factors. Pseudomonas aeruginosa, for example, is a pervasive opportunistic pathogen with numerous mechanisms of virulence, yet no described E3 ligases or deubiquitinases. We have developed a workflow to functionally screen natively secreted bacterial effectors for ubiquitin regulatory activities. After benchmarking this approach on Salmonella Typhimurium, Enteropathogenic Escherichia coli, and Shigella flexneri, we identified a cryptic E3 ligase secreted by P. aeruginosa, which we have termed Pseudomonas Ub ligase 1 (PUL-1). PUL-1 resembles none of the other E3 ligases previously established in or outside of the eukaryotic system. Importantly, in an animal model of P. aeruginosa infection, PUL-1 ligase activity plays an important role in regulating virulence. Thus, functional identification of ubiquitin-targeted effectors can expand our appreciation of ubiquitin regulation during bacterial pathogenesis.