Withaferin A and Withanolide A-mediated silver nanoparticles target bacterial motility, metabolism, and stress...

Biogenic silver nanoparticles (Bio-AgNPs) were synthesized using Withania somnifera root extract and evaluated for their antimicrobial and biosafety profiles. LC-MS confirmed key withanolides, including Withaferin A. The nanoparticles exhibited a UV-Vis peak at 446 nm, a crystalline core size of ∼15.65 nm, a hydrodynamic diameter of ∼134.85 nm, and a zeta potential of -25.29 mV. Bio-AgNPs showed potent antibacterial activity against Escherichia coli K12, with MICs between 156.25 and 312.5 ppm and superior efficacy to AgNO₃. RNA-seq analysis revealed downregulation of flagellar genes (flgD, flgG, fliC), maltose transporters (malK, malF, lamB), and citrate metabolism (citT, citD), indicating impaired motility and nutrient uptake. Upregulation of metal efflux (cusF, cusB) and electron transport (fixA, fixB) genes reflected oxidative stress response. Cytotoxicity assays on WI-38 and HEK-293 cells showed >80 % viability up to 156.25 ppm. These findings support Bio-AgNPs as biocompatible, plant-derived antimicrobials effective against Gram-negative bacteria.