Second-generation lysocins as therapeutics for treating <i>Pseudomonas aeruginosa</i> infections

<i>Pseudomonas aeruginosa</i> is a leading cause of nosocomial infections, including pneumonia and urinary tract infections, and the primary cause of morbidity and mortality in cystic fibrosis patients. The emergence of multidrug-resistant strains makes these infections life-threatening. To overcome this challenge, lysocins can be employed as novel antipseudomonals. Lysocins use components of the pyocin antimicrobial system to deliver bacteriophage lysins to their peptidoglycan substrate in <i>Pseudomonas</i>. Peptidoglycan cleavage causes membrane destabilization, cytoplasmic leakage, and disruption of the proton motive force, thereby killing the cell. In our previous proof-of-concept study, the PyS2-GN4 lysocin killed only one-third of <i>P. aeruginosa</i> strains due to the targeted receptor. This limitation can now be circumvented by engineering second-generation lysocins that bind and translocate through highly conserved <i>Pseudomonas</i>-specific receptors. One lysocin, PyS5-I-GN4, uses a single domain from pyocin S5 to deliver the GN4 lysin through the conserved ferric pyochelin transporter, consequently killing 95% of multidrug-resistant clinical isolates tested. Importantly, PyS5-I-GN4 displayed antibiofilm properties and was bactericidal in serum and lung surfactant. Serum inactivation observed for lysins is not seen for lysocins, making this approach more effective for treating systemic Gram-negative bacterial infections. Despite its broadened pseudomonal strain coverage, PyS5-I-GN4 demonstrated narrow-spectrum antibacterial activity toward <i>P. aeruginosa</i> only and lacked cytotoxicity toward human cells. A single dose of lysocin was protective and reduced bacteria multiple log₁₀-fold in the lungs and secondary organs in a neutropenic murine lung infection model. These findings support lysocins as therapeutics for <i>P. aeruginosa</i> and provide insight into designing future constructs for other Gram-negative pathogens.