An Interbacterial Cysteine Protease Toxin Inhibits Cell Growth by Targeting Type II DNA Topoisomerases GyrB and ParE

Bacteria secrete a diverse array of toxic effectors to antagonize competitors, profoundly influencing the composition of microbial communities. Previous studies have identified an interbacterial toxin predicted to exhibit proteolytic activity that is broadly distributed among Gram-negative bacteria. However, the precise mechanism of intoxication remains unresolved. Here, we demonstrate that one such protease toxin from Escherichia coli , Cpe1, disrupts DNA replication and segregation by recognizing and cleaving conserved sequences in the essential type II DNA topoisomerases GyrB and ParE. Cpe1 belongs to the papain-like cysteine protease family and is associated with toxin delivery pathways, including the type VI secretion system and contact-dependent growth inhibition. The crystal structure of Cpe1 in complex with its immunity protein reveals a neutralization mechanism involving competitive substrate binding rather than active site occlusion, distinguishing it from previously characterized effector-immunity pairs. Collectively, our findings unveil a unique mode of interbacterial intoxication and provide insights into how bacteria protect themselves from self-poisoning by protease toxins.