Disparate immunity proteins independently inactivate an antibacterial nuclease toxin

Bacteria secrete a diverse arsenal of protein toxins that inhibit the growth of competing species. To avoid self-intoxication, antibacterial toxins are encoded alongside cognate immunity proteins that typically bind to and occlude a toxin's active site, thus neutralizing its activity. Multiple immunity proteins rarely target the same toxin, reflecting the strong coevolution between toxins and their immunity factors. Here, we identify and characterize an antibacterial toxin in a clinical isolate of Pseudomonas aeruginosa, which we term Tde5, and find that it is inhibited by two structurally distinct immunity proteins, Tdi5a and Tdi5b. Informatic analyses reveal that Tde5 is a member of the ββα-metal DNase superfamily, and accordingly, we find that this enzyme inhibits bacterial growth by nonspecifically degrading DNA. This antibacterial activity is counteracted by either Tdi5a or Tdi5b, suggesting that these two immunity proteins function independently to protect bacteria against Tde5-mediated toxicity. Using biochemical and biophysical approaches, we show that Tdi5a and Tdi5b interact with Tde5 with sub-nanomolar affinities and that a trimeric complex forms between these three proteins, indicating that the two immunity factors bind to the toxin through nonoverlapping interfaces. A 1.73 Å X-ray crystal structure of the Tde5-Tdi5a complex reveals that Tdi5a binds to Tde5 through a large electrostatic interface and occludes the toxin's active site, whereas structural modeling reveals that Tdi5b binds an exosite distal to the toxin's active site. Together, these findings define a previously unrecognized dual-immunity mechanism and expand our understanding of antibacterial toxin-immunity dynamics, informing future studies of interbacterial competition and virulence.