Oka, G. U.; Adan, W. C.; dos Santos, T. R.; Cenens, W.; Souza, D. P.; Farah, C. S.

Bacteria can outcompete rivals by using specialized secretion systems to deliver toxic effectors into prey cells. How these systems distinguish prey from kin remains a fundamental unanswered question. In many Xanthomonadales (Lysobacterales) species, the antibacterial type IV secretion system (X-T4SS) mediates cell-cell contact-dependent killing of competing bacteria. Here, we identified XAC2611, a chromosomally encoded, cysteine-rich DUF4189 protein, as essential for preventing X-T4SS-mediated fratricide among sibling cells in Xanthomonas citri. XAC2611 homologs are found within or proximal to the genomic loci encoding almost all identified X-T4SSs. Live-cell microscopy and biochemical data reveal that XAC2611 is abundantly produced, widely distributed throughout the periplasm of recipient cells, and required to prevent X. citri cells from intoxicating each other in a contact-dependent manner. We also show that XAC2611 homologs from Stenotrophomonas maltophilia protect X. citri cells from attack by S. maltophilia. Protein-protein interaction assays show that XAC2611 interacts directly with the VirB5 subunit. Since VirB5 is predicted to localize at the tip of the X-T4SS pilus, its interaction with XAC2611 in a neighboring sister cell could block X-T4SS-mediated effector delivery. We therefore name this family of proteins trans-intoxication protection factors (Tpfs).