The role of a phage defense island in enterohemorrhagic Escherichia coli

Introduction: Enterohemorrhagic Escherichia coli (EHEC) O175:H7 strain EDL933 is a foodborne pathogen that can cause life-threatening infections, such as bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). We found that EDL933 is highly resistant to most phages that efficiently lyse E. coli K12 strains. Using DefenseFinder, three nuclease/helicase-based phage defense systems were predicted to be encoded on an O-island in EHEC strains, including Zorya-II, Druantia-III and Serket-I. Goals: We hypothesize that this phage defense island contributes to the strong phage resistance of EHEC and that the three systems act synergistically in the anti-phage defense. Thus, we aimed to determine the phage spectrum protected by each defense system and to study their synergistic interactions. We are further interested in the molecular mechanisms of the anti-phage defense. Materials & Methods: We used EOP and growth assays under phage infections to analyze the phage spectrum and synergy of the defense systems. Biochemical and structural analyses were applied to study the molecular mechanism of the Druantia-III and Serket-I antiphage defense. Results: EOP assays with druHE, zorABE and serABCD-deficient strains revealed that the three defense systems protect to different extents against a broad spectrum of restriction-sensitive phages, but not against hypermodified Tevenvirinae. Using double system mutants, we found that Zorya-II, Druantia-III and Serket-I provide additive resistance and synergy towards different phages. Biochemical and structural analysis established that DruE functions as 3"-5"-directional DNA helicase and nuclease containing unique molecular tools, which affect its molecular activities. Using in vivo complementation studies with DruE variants, we established that the DUF1998 domain with its conserved 4Cys-Zn2+ cluster is essential for the anti-phage defense, while the PLD1 and PLD2 domains are dispensable for functionality. Summary: Overall, we hypothesize that the three defense systems cooperate in phage DNA sensing, discriminate phage and host DNA by methylation, cleave non-methylated phage DNA at specific restriction sites and subsequently degrade phage DNA.