The co-localizing Zorya II, Druantia III, and ARMADA II defense systems on O-island 172 confer synergistic anti-phage defense in enterohemorrhagic Escherichia coli

In this study, we show that the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 Δ stx1/2 is highly resistant to phages. The genes encoding the three phage defense systems Zorya II, Druantia III, and ARMADA II were found to co-localize on O-island 172 (OI-172) in EDL933. The three phage defense systems co-occur in 426 E. coli strains comprising 66 distinct serotypes, including 277 O157:H7 isolates and 117 diverse non-pathogenic strains. Efficiency of plating (EOP) assays were used to explore the phage spectrum and synergy of the three defense systems and their contribution to the phage resistance of strain EDL933 Δ stx1/2 . Using Δ zorABE, Δ druHE , and Δ armABCD single system deletion mutants in EDL933 Δ stx1/2 , we showed that Druantia III and ARMADA II protect to different extents against a broad spectrum of phages, including the Drexlerviridae , Siphoviridae , Demerecviridae , Vequintavirinae and Autographiviridae , but not against hypermodified Tevenvirinae . Additionally, Zorya II caused strong protection only against Autographiviridae . Furthermore, EOP assays of combined system mutants revealed strong synergistic interactions of Druantia III and ARMADA II to provide more robust immunity against a similar phage spectrum than the additive protection of the individual systems. In contrast, Druantia III and Zorya II act only weakly synergistically against a few phages. Altogether, our results revealed that Druantia III and ARMADA II are responsible for most of the phage resistance of EDL933 Δ stx1/2 , whereas Zorya II provides additional immunity against podoviruses. Future studies are underway to elucidate the molecular basis of the synergistic interactions between the Druantia III and ARMADA II systems.