Bacterial manipulation of atypical ubiquitin signaling - Project Summary Post-translational ubiquitin signaling is emerging as a key component of both immune defense as well as bacterial virulence. Though they lack a canonical ubiquitin system of their own, pathogenic bacteria have evolved secreted `effector' proteins to manipulate ubiquitin signals, thereby gaining access to host cell processes including targeted protein degradation, trafficking, and inflammation. The far-reaching importance of ubiquitin signaling across many cellular processes stems from its ability to form a diverse set of polymeric chains that signal for distinct outcomes. The complexity of ubiquitin signaling vastly outweighs our understanding of its regulation and cellular outcomes. While the signaling roles for some types of polyubiquitin are known (e.g., protein degradation directed by Lys48-linked polyubiquitin), the functions of many so-called `atypical' subtypes have remained a mystery despite decades of research. Remarkably, we and others have recently identified a series of bacterial effector proteins that have evolutionarily converged upon the specific regulation of atypical polyubiquitin signals linked through Lys6, suggesting this post-translational signal must play a central role within the host-pathogen interface. In a fascinating paradox, we find that bacterial pathogens like enterohemorrhagic Escherichia coli (EHEC) have evolved to upregulate Lys6 polyubiquitin, while others such as Legionella pneumophila have evolved to downregulate it. Based on our preliminary data and clues from outside the context of infection, we propose that bacteria hijack Lys6 polyubiquitin as an accelerated proteasomal degradation signal. We propose to test this hypothesis from three perspectives: 1) understanding the molecular basis of Lys6 polyubiquitin specificity, 2) defining the roles of bacterial E3 ligases that form Lys6 polyubiquitin signals during infection, and 3) identifying the molecular pathways restricted by bacterial deubiquitinases that remove Lys6 signals. Our study benefits from multiple conceptual and technical innovations, including the ability to rewire bacterial E3 ligases between Lys6 and Lys48 specificity, allowing detailed studies of their effects on protein degradation and bacterial virulence. In addition, by removing the ability of L. pneumophila to restrict Lys6 polyubiquitination, for the first time we can identify upstream and downstream regulators of Lys6 signals, as part of what could be a new arm of cell-autonomous immunity. The insights this work will generate for the fields of bacterial pathogenesis and post-translational signaling are highly significant, and could lead to new opportunities for anti-virulence therapeutics. Over the next five years we will open a window into ubiquitin signaling at the host- pathogen interface in order to understand the evolutionary benefits of regulating this poorly studied, atypical Lys6 polyubiquitin signal.
