Sequencing of pTEF2-resistant Enterococcus Phage 47

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of new antibiotics needed to combat these infections remains stagnant. MDR enterococci, which are a common cause of hospital-acquired infections, are emerging as one of the major contributors to this crisis. An alternative therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which entails the use of lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of phage resistance and the mechanisms underlying this resistance are unknown. Here, we use a CRISPRi screen to identify a genetic locus found on a mobilizable plasmid from vancomycin-resistant Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) and we show that this enzyme is sufficient to restrict the replication of the lytic phage in E. faecalis. We further find that phages can evolve to overcome restriction by acquiring a conserved mutation in a novel TIV-RE inhibitor protein, which we have named arfA (anti-restriction-factor A). ArfA binds and inactivates a diverse array of TIV-REs. Overall, our findings significantly advance our understanding of phage defense in E. faecalis and provide mechanistic insight into how phages can evolve to overcome antiphage defense systems.