A novel CXCL8(3–72) K11R/G31P–BPI 233 hybrid protein modulates neutrophil-driven inflammation and protects against carbapenem-resistant Acinetobacter baumannii infection

Carbapenem-resistant Gram-negative bacteria (CRGNB) pose a severe global health threat. Carbapenem-resistant Acinetobacter baumannii (CRAB) is one of the most critical threats among CRGNB, and effective non-antibiotic therapeutic strategies are urgently needed. Bactericidal/permeability-increasing protein (BPI)-derived molecules display antibacterial and endotoxin-neutralizing activities against Gram-negative bacteria, whereas CXCL8(3–72) K11R/G31P (G31P), an analog of CXCL8 with the mutations of CXCL8 at K11 and G31 positions, could antagonize ELR-CXC chemokine-mediated neutrophil chemotaxis. Here, we engineered a dual-function fusion protein, G31P–BPI233, to combine BPI-mediated antibacterial activity with modulation of neutrophil-driven inflammation. Recombinant G31P–BPI233 was expressed in CHO-K1 cells and characterized through safety evaluation, endotoxin neutralization, bactericidal, serum killing, neutrophil killing, chemotaxis, cytokine, phagocytosis, and NET-associated marker assays, followed by validation in mouse models of CRAB septicemia and pneumonia. Short-term safety assessment suggested preliminary tolerability over 24 h, while G31P–BPI233 reduced residual endotoxin activity and exhibited concentration-dependent bactericidal activity against multiple Gram-negative bacteria. At molar-matched doses, it retained G31P-mediated inhibition of neutrophil migration and BPI233-associated bactericidal and cytokine-suppressive activities. In CRAB models, G31P–BPI233 improved septicemia survival and reduced pneumonia-associated bacterial burden and inflammatory injury, with endpoint-specific advantages over G31P, BPI233, or their equimolar mixture. Several pneumonia outcomes did not differ significantly between G31P–BPI233 and colistin. Mechanistic studies using BPI-deficient mice further demonstrated that endogenous BPI contributes to bacterial clearance, endotoxin control, and neutrophil maturation and effector function during CRAB infection. Collectively, these findings identify G31P–BPI233 as a promising host-directed antibacterial fusion protein for severe CRAB infection, with potential translational implications for the broader management of CRGNB.