Contributors: Institut de biologie structurale (IBS - UMR 5075); Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG); Direction de Recherche Fondamentale (CEA) (DRF (CEA)); Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)); Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA); Centre d’Etude des Pathologies Respiratoires (CEPR), UMR 1100 (CEPR); Université de Tours (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM); Centre Hospitalier Universitaire CHU Grenoble (CHUGA); ANR-22-CE18-0009,HUMABACT,Anticorps monoclonaux humains thérapeutiques contre les bactéries multirésistantes par identification de cibles, isolement d'anticorps à partir de cellules B uniques et biologie structurale(2022); ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010); ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010); ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017)
نبذة مختصرة : Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting to isolate, from cystic fibrosis patients, human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. Further, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium . The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa -infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for developing effective alternatives to traditional antibiotics in the fight against this resilient pathogen.
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