Identification of Secreted Copper-Binding Peptides Associated with Virulence in Pathogenic Pseudomonas and Bordetella

Peptides that contain 5-thiooxazole post-translational modifications have recently been identified from various bacteria and implicated in copper homeostasis and virulence. However, knowledge of their copper-binding affinities is limited, and their roles in virulence are not well understood. In this work, we discovered three new 5-thiooxazole-containing peptides from two opportunistic human pathogens, Pseudomonas aeruginosa and Bordetella bronchiseptica. We heterologously produced and structurally characterized two different peptides that originate from P. aeruginosa, pseudomonin and pseudomonin-efh, the latter of which also contains two EF-hand domains for calcium binding. We show that 5-thiooxazole modification in both peptides enables strong Cu(I) binding at 10–16–10–17 M affinity and weaker Cu(II) binding at 10–7 M affinity. Pseudomonin-efh undergoes calcium-dependent changes of overall secondary structure that are negated by copper. Genetic studies revealed the 5-thiooxazole modification of pseudomonin-efh is required for calcium-dependent virulence regulation in a lettuce infection model for P. aeruginosa. Further, we identified and characterized a related 5-thiooxazole-containing peptide, bordetellin, secreted by its native producer, B. bronchiseptica, in the virulent phase. Our work demonstrates a strong preference for Cu(I) binding by 5-thiooxazole-containing peptides and uncovers a role of these peptides in Ca/Cu-dependent virulence regulation. Identifying 5-thiooxazole-containing peptides from pathogenic bacteria and detecting their secretion in the native producer is a critical step toward understanding the functions of these peptides in virulence and pathogen–host interactions.