The Potential Link between Transmembrane Sensors and Nucleic Acid Remodeling in the Shango Anti-Phage Defense System

ongoing

The SHARP (Shango Anti-phage Remodeling Pathway) project aims to explore the molecular mechanism of Shango system, a widespread bacterial anti-phage defense module. While the well-known CRISPR-Cas systems have revolutionized genome editing, bacterial defense strategies encompass over 130 distinct gene modules and present a treasure chest for novel biotechnologies. To eliminate viral threats, these systems employ various mechanisms, either pre- or post-injection. Shango consists of three core genes: SngA, SngB, and SngC with bioinformatically predicted functions thus far. SngA, annotated as tellurite stress response element, has two transmembrane helices and is proposed to detect an unknown viral signal. Its C-terminal domain may recruit SngB and SngC. SngB, a member of the AAA+ ATPase family, together with SngC, a DEAD-box helicase, likely participates in DNA remodeling. In this project, I hypothesize that Shango induces changes in nucleic acid topology, thus impairing phage genome integrity and preventing its replication, though the exact mechanism remains elusive. By isolating phage escapers capable of bypassing Shango, I aim to identify the triggering signal. Subsequently, I will analyze the specificity and unwinding activity of SngB and SngC using biochemical assays. Advanced techniques such as single-particle cryogenic electron microscopy (cryo-EM) will be used to elucidate distinct functional states of Shango. Leveraging Dr. N. Taylor’s expertise, I aim to assemble the complete complex of Shango together with its transmembrane helices. To reveal the spatial-temporal regulation of the system during phage attack, I will employ Total Internal Reflection Fluorescence (TIRF) microscopy to visualize the co-localization of Shango proteins and phages. This project not only explores new aspects of bacterial stress responses, but also aims to pioneer tools for signal-induced nucleic acid manipulations, opening up the possibilities in biotechnological applications.