Zhang Yu, Guo Shiyu, Jiang Liwen, Wang Siqi, Hou Weitong, Qiu Xiran, Shen Hui, An Maomao
Department of Pharmacology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, People's Republic of China.
Department of Pharmacy, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
Antimicrob Agents Chemother. 2025 Oct;69(10):e0040525. doi: 10.1128/aac.00405-25. Epub 2025 Aug 18.
bloodstream infections carry mortality rates exceeding 60%, with escalating antibiotic resistance dramatically limiting therapeutic options. The type III secretion system (T3SS), a virulence apparatus delivering cytotoxic effectors via PcrV-dependent translocation pores, represents a therapeutic target. Here, we developed a monoclonal antibody (5C8) targeting the central domain (H106-D173) of PcrV, which regulates translocation pore size. 5C8 demonstrated sub-nanomolar affinity (KD = 0.32 nM) via biolayer interferometry and broad neutralization efficacy against clinical isolates (IC: 0.32-1.47 μg/mL). In murine bloodstream infection models, 5C8 conferred improved survival against cytotoxic () and invasive () strains ( < 0.01 vs controls), reducing bacterial loads in lungs/kidneys by 1.5-log colony-forming unit ( < 0.01) and suppressing interleukin-6 levels by 60-82% ( < 0.01). Mechanistic studies revealed 5C8's dual action: blocking effector release (ExoU/ExoT reduced by 41-88% via liquid chromatography-mass spectrometry) and constricting T3SS pores below 1.2 nm (carbohydrate exclusion assay). Molecular docking identified D125/K129/Y145 as critical binding residues, validated by alanine scanning and mutant construction. Humanized Hu5C8 retained potency (KD = 0.55 nM) with extended half-life (t = 91.26 h) through Fc receptor engineering. As an inhibitor targeting the pore size-determining domain of PcrV, 5C8 disrupts virulence through a novel dual mechanism, providing a paradigm-shifting strategy against multidrug-resistant , bridging a critical gap in sepsis management.
血流感染的死亡率超过60%，抗生素耐药性不断上升，极大地限制了治疗选择。III型分泌系统（T3SS）是一种通过依赖PcrV的转运孔递送细胞毒性效应蛋白的毒力装置，是一个治疗靶点。在此，我们开发了一种靶向PcrV中央结构域（H106-D173）的单克隆抗体（5C8），该结构域调节转运孔大小。5C8通过生物层干涉术显示出亚纳摩尔亲和力（KD = 0.32 nM），对临床分离株具有广泛的中和效力（IC：0.32-1.47 μg/mL）。在小鼠血流感染模型中，5C8提高了对细胞毒性（）和侵袭性（）菌株的存活率（与对照组相比P<0.01），使肺/肾中的细菌载量降低1.5个对数集落形成单位（P<0.01），并将白细胞介素-6水平抑制60-82%（P<0.01）。机制研究揭示了5C8的双重作用：阻断效应蛋白释放（通过液相色谱-质谱法，ExoU/ExoT减少41-88%）和将T3SS孔收缩至1.2 nm以下（碳水化合物排斥试验）。分子对接确定D125/K129/Y145为关键结合残基，通过丙氨酸扫描和突变体构建得到验证。通过Fc受体工程，人源化Hu5C8保留了效力（KD = 0.55 nM），半衰期延长（t = 91.26 h）。作为一种靶向PcrV孔径决定结构域的抑制剂，5C8通过一种新的双重机制破坏毒力，为对抗多重耐药菌提供了一种范式转变策略，填补了脓毒症管理中的关键空白。
