YtnP lactonase from Bacillus licheniformis T2 mitigates virulence and biofilm formation in carbapenem-resistant Acinetobacter baumannii via quorum sensing quenching
Abstract
Carbapenem-resistant Acinetobacter baumannii (CRAB) has become a predominant nosocomial pathogen worldwide, with biofilm formation and quorum sensing (QS) primarily responsible for its high antimicrobial tolerance and clinical refractory infections. Targeting bacterial virulence rather than conventional bactericidal strategies represents a promising alternative to combat multidrug-resistant CRAB. In this study, we isolated a N-acyl homoserine lactonase YtnP from mangrove rhizosphere-derived Bacillus licheniformis T2. We comprehensively biochemically characterized the YtnP and systematically validated its potent quorum-quenching, antibiofilm and anti-virulence activities against clinical CRAB strains both in vitro and in vivo model. YtnP is a 280-amino-acid metalloenzyme of approximately 30 kDa containing a conserved HXHXDH motif, exhibiting optimal activity at 37°C –45°C and pH 7.0–7.5. Distinct from classical lactonases, YtnP maintained high catalytic activity without exogenous Zn 2+ supplementation but was markedly inhibited by EDTA and excess Zn 2+ . Functional assays revealed that YtnP efficiently degraded the key QS signal 3-OH-C12-HSL with a degradation rate of 59.5% without affecting the planktonic growth of CRAB. YtnP significantly dismantled mature CRAB biofilms, reducing biofilm biomass by 55.56% and maximum thickness by 61.76%, and prominently downregulated the transcription of virulence-related outer membrane protein genes ompA and csuA/B by nearly 50% and 52.6%, respectively. In the Galleria mellonella model, YtnP remarkably alleviated CRAB metabolite-induced lethality and increased the 36-h larval survival rate nearly sixfold. Collectively, mangrove-derived YtnP acts as a potent quorum-quenching enzyme that suppresses CRAB biofilm formation and virulence in a non-bactericidal manner. This work highlights the potential of YtnP as a promising anti-virulence adjuvant candidate for the development of alternative therapeutic strategies against intractable CRAB infections.