RESUMEN
The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses 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 resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.
Asunto(s)
Bacteriófagos , Enterococcus faecalis , Proteínas Virales , Enterococcus faecalis/virología , Enterococcus faecalis/genética , Bacteriófagos/genética , Bacteriófagos/fisiología , Proteínas Virales/metabolismo , Proteínas Virales/genética , Enzimas de Restricción del ADN/metabolismo , Enzimas de Restricción del ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Plásmidos/genética , Enterococos Resistentes a la Vancomicina/genética , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genéticaRESUMEN
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. A potential 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 CRISPR interference (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 missense mutation in a novel TIV-RE inhibitor protein encoded by many enterococcal phages. We show that this inhibitor, which we have named anti-restriction-factor A (arfA), directly binds to and inactivates diverse TIV-REs. Overall, our findings significantly advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages can evolve to overcome antiphage defense systems.