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These hits were validated in comparative studies with V. parahaemolyticus wild type and a mutant producing an inactive VopS (H348A). The method further allowed to decipher the sites of modification and facilitated a time-dependent analysis of AMPylation during infection. #link# Overall, the methodology provides a reliable detection of host AMPylation in situ and thus a versatile tool in monitoring infection processes.Antimicrobial peptides are promising alternatives to traditional antibiotics. A group of self-assembling lipopeptides wa