NV716 acts as an envelope-active adjuvant that enhances antibiotic accumulation in Pseudomonas aeruginosa.

Treatment of Gram-negative infections remains challenging due to limited compound penetration and the presence of efflux mechanisms. Pseudomonas aeruginosa, in particular, exhibits strong intrinsic resistance, leaving few effective therapeutic options. Here, we provide mechanistic insight into the activity of NV716, a polyaminoisoprenyl antibiotic adjuvant previously described in Gram-negative bacteria, by linking outer membrane perturbation to quantitative changes in intracellular antibiotic accumulation. In P. aeruginosa and Escherichia coli, NV716 increased intracellular antibiotic accumulation and potentiated selected antibiotics, with the most pronounced effects observed for doxycycline. Studies using efflux-deficient and porin-mutant strains indicate that NV716 perturbs outer membrane organization, thereby facilitating enhanced intracellular antibiotic accumulation. Population-scale assays revealed increased outer membrane vesicle (OMV) release, and high-resolution imaging visualized membrane-associated alterations and OMV formation. Truncation of the lipopolysaccharide core sensitized P. aeruginosa to NV716, consistent with increased accessibility of lipid A. Together, these findings establish a quantitative framework that links controlled outer membrane perturbation to intracellular antibiotic accumulation and antibiotic-class-dependent potentiation.