Mechanistic blockade of type III secretion by a monoclonal antibody targeting the pore size-determining domain of PcrV.

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.