Table_1_Antibodies Against Pseudomonas aeruginosa Alkaline Protease Directly Enhance Disruption of Neutrophil Extracellular Traps Mediated by This Enzyme.docx

Extracellular traps released by neutrophils (NETs) are essential for the clearance of Pseudomonas aeruginosa. Alkaline protease (AprA) secreted by P. aeruginosa negatively correlates with clinical improvement. Moreover, anti-AprA in patients with cystic fibrosis (CF) can help identify patients with aggressive forms of chronic infection. However, the mechanism underlying the clinical outcomes remains unclear. We demonstrated that aprA deficiency in P. aeruginosa decreased the bacterial burden and reduced lung infection. AprA degraded NET components in vitro and in vivo but did not affect NET formation. Importantly, antibodies induced by AprA acted as an agonist and directly enhanced the degrading activities of AprA. Moreover, antisera from patients with P. aeruginosa infection exhibited antibody-dependent enhancement (ADE) similar to that of the antibodies we prepared. Our further investigations showed that the interaction between AprA and the specific antibodies might make the enzyme active sites better exposed, and subsequently enhance the recognition of substrates and accelerate the degradation. Our findings revealed that AprA secreted by P. aeruginosa may aggravate infection by destroying formed NETs, an effect that was further enhanced by its antibodies.

中性粒细胞释放的胞外陷阱（Neutrophil Extracellular Traps，NETs）对于清除铜绿假单胞菌至关重要。铜绿假单胞菌分泌的碱性蛋白酶（Alkaline Protease, AprA）与临床改善程度呈负相关。此外，囊性纤维化（Cystic Fibrosis, CF）患者体内的抗AprA抗体可辅助识别罹患侵袭性慢性感染的患者。然而，其介导临床结局的具体机制仍未阐明。本研究证实，铜绿假单胞菌中aprA基因缺失可降低细菌载量并减轻肺部感染。AprA可在体外及体内降解NET组分，但不影响NET的形成过程。尤为关键的是，AprA诱导产生的抗体可作为激动剂，直接增强AprA的降解活性。此外，铜绿假单胞菌感染患者的血清所展现出的抗体依赖增强效应（Antibody-Dependent Enhancement, ADE），与本研究制备的抗体效果一致。进一步研究表明，AprA与其特异性抗体的结合可使该酶的活性中心更好地暴露，进而增强底物识别效率并加速降解进程。本研究结果揭示，铜绿假单胞菌分泌的AprA可通过破坏已形成的NETs加重感染，而其诱导产生的抗体可进一步强化这一致病效应。