Himpich, S. | bioRxiv Science

bioRxiv2026

A compact Druantia defense clears phage infections via single-stranded DNA recognition and directional duplex unwinding

Bacteria encode diverse anti-phage defense systems triggered by invader-specific molecular cues. Here, we report that the compact type III-A Druantia system recognizes exposed single-stranded DNA to drive phage clearance. Using representative systems from Escherichia coli, we show that the two encoded proteins, DruE and DruH, together clear restriction-sensitive or recombination-prone phages without affecting cell growth or viability. DruE dimerizes and engages DNA at exposed single-stranded regions to unwind DNA with 3'-to-5' directionality, resorting to unique molecular lock, wedge, and clamp elements that aid strand separation and processive translocation. DruH is a monomer in isolation and indirectly interacts with DruE and other host proteins under uninfected conditions, with an infection resulting in the dissociation of the complex. Taken together, our results reveal that exposed single-stranded DNA can trigger bacterial immunity through the directional helicase activity of type III-A Druantia. HighlightsO_LIThe E. coli Druantia III-A defense, comprising DruE and DruH, clears infecting phages C_LIO_LIDruE dimers bind exposed single-stranded DNA and unwind the upstream DNA duplex C_LIO_LI3'-to-5' DNA unwinding is aided by molecular lock, wedge, and clamp elements C_LIO_LIDruE interacts with DruH and host proteins, which are displaced upon infection C_LI

microbiology↗

bioRxiv2025

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 {Delta}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 {Delta}stx1/2. Using {Delta}zorABE, {Delta}druHE, and {Delta}armABCD single system deletion mutants in EDL933 {Delta}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 {Delta}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 defense systems. IMPORTANCEShiga toxin-producing E. coli (STEC) O157:H7 strains cause life-threatening diseases, such as hemorrhagic colitis and the hemolytic uremic syndrome. Currently, EHEC infections can be only treated symptomatically, since antibiotics are not recommended due to induction of the Shiga toxin. While phage therapy could offer a treatment option, we show that the EHEC strain EDL933 {Delta}stx1/2 is highly resistant to many phages. EOP assays of defense mutants in the host strain revealed that the co-occurring defense systems Zorya II, Druantia III, and ARMADA II on OI-172 confer most of the phage resistance in EHEC. Moreover, our data uncovered that Druantia III and ARMADA II act synergistically in the anti-phage defense in EDL933 {Delta}stx1/2, explaining the robust immunity against a broad spectrum of phages. These results support the idea that the design of novel inhibitors against the Druantia III and ARMADA II systems could be combined with phage therapies to efficiently eradicate EHEC.

microbiology↗