DOI: 10.1128/mbio.00943-26 ISSN: 2150-7511
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
Huihui Li, Vu Van Loi, Costanza Borelli, Stephanie Himpich, Michaela Projahn, Lena M. Grass, Thi Phuong Thao Nguyen, Markus C. Wahl, Haike Antelmann ABSTRACT
In this study, we found that the enterohemorrhagic
Escherichia coli
(EHEC) strain EDL933 Δ
stx1/2
is highly resistant to many phages. The three phage defense systems Zorya II, Druantia III, and DIS
ARM
-related
A
ntiviral
D
efense
A
rray (ARMADA II) are encoded on O-island 172 (OI-172) in strain EDL933. Sequence comparison revealed that the three phage defense systems co-occur in 426
E. coli
strains, including 277 O157:H7 isolates and 117 diverse non-pathogenic strains. Efficiency of plating (EOP) assays was used to explore the phage spectra and synergy of the three defense systems, and their contribution to the phage resistance of the host 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 significantly protect to different extents against a similar spectrum of phages, including the
Drexlerviridae
,
Queuovirinae
,
Demerecviridae
,
Vequintavirinae
,
Stephanstirmvirinae
, and
Autographivirales
, whereas Zorya II only provided protection against
Autographivirales
. Further EOP and growth experiments indicated significantly synergistic and additive interactions of Druantia III and ARMADA II against a broad phage spectrum. The defense pair Druantia III and Zorya II was found to interact additively 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
Autographivirales
. Future studies are underway to elucidate the molecular basis of the synergistic interactions between the Druantia III and ARMADA II defense systems.
IMPORTANCE
Shiga toxin-producing
Escherichia coli
strains cause life-threatening diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. Currently, enterohemorrhagic
E. coli
(EHEC) infections can only be treated symptomatically, since antibiotics are not recommended due to induction of the Shiga toxin. While phage therapy could offer a treatment option, we found that the EHEC strain EDL933 Δ
stx1/2
is highly resistant to many phages. Efficiency of plating (EOP) assays of defense mutants in the host strain revealed that the co-occurring defense systems Zorya II, Druantia III, and DIS
ARM
-related
A
ntiviral
D
efense
A
rray (ARMADA II) on O-island 172 (OI-172) confer most of the phage resistance in EHEC. Moreover, Druantia III and ARMADA II were shown to interact additively and synergistically in the anti-phage defense in EDL933 Δ
stx1/2
, providing a more robust immunity than each system alone. These results support the idea that the discovery of inhibitors or anti-defense proteins against the Druantia III and ARMADA II systems could be combined with phage therapies to efficiently eradicate EHEC.