Deciphering the broad host range of Kayvirus phages infecting Staphylococcus aureus through investigation of anti-defense strategies

Phages of the genus Kayvirus hold significant promise for phage therapy due to their broad host range, enabling them to infect numerous strains of the nosocomial and veterinary pathogen Staphylococcus aureus, a major threat as antibiotics become increasingly ineffective. This broad host range is primarily attributed to their ability to recognize conserved structures in the S. aureus cell wall, facilitating attachment to diverse strains. However, after DNA injection into the host cytoplasm, diverse intracellular defense systems can hinder the phage replication cycle. This indicates that Kayvirus phages have evolved mechanisms to evade S. aureus immune defenses, ensuring successful infection. This project aims to uncover how Kayvirus phages overcome these intracellular defenses, focusing on viral proteins that trigger host defense activation and anti-defense proteins that inhibit these systems. In the first work package (WP1), a S. aureus library harboring plasmid- encoded defense systems will be challenged with phages to isolate escape mutants. Whole genome sequencing of these mutants will pinpoint key mutated genes, and subsequent validation will confirm their role as triggers. Comparative genomics will then elucidate how trigger mutations or their absence enable Kayvirus phages to bypass host defense systems. WP2 will focus on identifying novel anti-defense proteins by selecting candidates from early-expressed phage genes through bioinformatic analysis, cloning them onto inducible plasmids, and testing their ability to restore the infectivity of phages whose intracellular replication is blocked. WP3 will focus on characterizing these new anti-defense proteins, particularly their mode of action, using structural and biochemical approaches. By elucidating the underlying anti-defense mechanisms, this project will enhance our understanding of S. aureus phage-host interactions and provide valuable insights for enhancing staphylococcal phages therapeutic potential.