Molecular mechanisms of bacterial immune signaling through DNA damage

点击翻译按钮获取中文摘要

PROJECT SUMMARY Molecular mechanisms of bacterial immune signaling through DNA damage The availability of tens of thousands of bacterial genome sequences, plus new bioinformatics tools and new understanding of bacterial genome organization, has enabled the discovery and experimental characterization of dozens of anti-bacteriophage and anti-plasmid defense systems in bacteria. Since a typical bacterial genome encodes 3-6 distinct defense systems, a key question is whether and how these systems can coordinate their activities to synergistically fight an infection. In prior work on the widespread and diverse CBASS (Cyclic oligonucleotide-Based Anti-phage Signaling System) defense systems, we identified two transcriptional regulators – CapW and the two-protein CapH+CapP system – that boost CBASS gene expression in response to DNA damage. Together, CapW and CapH+CapP are associated with ~10% of CBASS systems, and are also found adjacent to a broad range of known and predicted bacterial defense systems including Pycsar, DISARM, and BREX. These findings suggest that CapW and CapH+CapP may mediate activation of antiviral defense in response to a universal signal of cell stress, DNA damage. Here, I will first identify the small-molecule or nucleic acid ligand that binds and activates CapW upon DNA damage. I will combine biochemical assays for CapW binding to both its target DNA and its ligand with x-ray crystallography to characterize the conformational changes imposed by the ligand to control CapW-DNA binding. This work will establish a mechanism for CapW, a widespread bacterial transcription factor. Next, I will test the idea that CapW and CapH+CapP mediate cooperation between antiviral defense systems by sensing DNA damage. Specifically, we hypothesize that DNA-targeting immune systems like restriction-modification and CRISPR-Cas create DNA damage that is sensed by CapW or CapH+CapP to activate a secondary defense system (CBASS or others) to reinforce the defensive response. I will systematically test this model by infecting cells encoding both a restriction-modification system and a CapW- or CapH+CapP-associated CBASS system to determine if the combination of these systems yields synergistic antiviral immunity. Additionally, I will test whether DNA damage sensing plays a role in defense-system synergy, using structure-based mutations to either CapW or CapP that eliminate DNA damage sensing. Together, these experiments will reveal the molecular mechanism of CapW, and the role of DNA damage sensors in mediating synergy in bacterial defense systems. The findings have the potential to establish a new paradigm in which DNA targeting defense systems constitute a first line of antiviral defense, and DNA damage-activated systems constitute a second line of defense with orthogonal mechanisms. Thus, instead of viewing bacterial defense systems in isolation, this work will establish how they cooperate to compose a comprehensive bacterial “immune system”.