Impact of CRISPR-associated transposons on anti-phage immunity in Vibrio cholerae

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PROJECT SUMMARY Vibrio cholerae is the causative agent of the infectious diarrheal disease, cholera, which affects several million individuals and causes ~100,000 deaths, annually. It has become increasingly clear in recent years that horizontal gene transfer events played a crucial role in the explosive diversification of a non-pathogenic strain from the Middle East into the present-day pathogenic El Tor biotype strain. Virulence and antibiotic resistance genes are broadly disseminated within marine Vibrio communities by mobile genetic elements (MGEs), including bacterial viruses, plasmids, and transposons, many of which permanently integrate their genetic payloads into the genome. Furthermore, dynamic interactions between V. cholerae and viruses directly impact the duration and severity of cholera outbreaks, and are potently influenced by the complex repertoire of antiviral defense systems spread by MGEs. Thus, viral predation and viral immunity affect V. cholerae fitness and pathogenicity, highlighting the need to better understand horizontal gene transfer processes that modulate antiviral defense. Our laboratory recently discovered a new class of transposable elements that encode nuclease-deficient CRISPR-Cas systems and spread via RNA-guided DNA integration, representing the first example of a fully programmable transposase. These CRISPR-transposon (CRISPR-Tn) systems are prevalent in Vibrio species, and our studies have thus far focused on a representative transposon derived from a clinical V. cholerae isolate sampled during the 2010 Haiti cholera epidemic. Remarkably, during our recent analyses of the genetic cargos found within a larger set of CRISPR-transposons, we uncovered a striking enrichment in antiviral defense genes, suggesting that these MGEs spread horizontally via conjugative plasmids and benefit host cells by mobilizing a rich complement of innate immune systems. Our central vision is to determine how V. cholerae immunity and pathogenicity is influenced by the acquisition of CRISPR-Tn cargo genes, while also developing RNA-guided transposases as a new tool for kilobase-scale genome engineering in V. cholerae. In Aim 1, we will employ bioinformatics, genetics, and high-throughput sequencing to comprehensively investigate the evolutionary and mechanistic diversity of CRISPR-Tn systems, and leverage the most active systems for high-efficiency genomic insertions and deletions in V. cholerae. In Aim 2, we will analyze the complete repertoire of CRISPR-Tn cargo genes and determine which gene clusters provide protection against Vibrio-specific viruses. Beyond shedding light broadly on the function of transposons in Vibrio, this proposal will expand our understanding of how MGEs promote rapid turnover of defense systems within bacterial populations as part of the pan-immune system. This topic is of increasingly critical importance, given the spread of antibiotic resistance genes and renewed interest in phage therapy for V. cholerae and numerous other pathogenic microorganisms.