Phosphate Limitation Modulates Vibrio cholerae Outer Membrane Vesicle Formation, Composition and Toxicity

Vibrio cholerae inhabits phosphorus-poor aquatic environments and host intestine, where it expresses genes regulated by the PhoB/PhoR two-component system in response to inorganic phosphate (Pi) limitation. Like other Gram-negative bacteria, V. cholerae releases outer membrane vesicles (OMVs), which carry proteins, lipids, and nucleic acids that contribute to adaptation, survival, and pathogenesis. Here, we investigated how Pi availability affects OMV production, composition, and toxicity in the pandemic strain N16961 and its {Delta}phoB mutant. Using transmission electron microscopy, atomic force microscopy, and nanoparticle tracking analysis, we show that OMV size remains constant ([~]140 nm) across conditions, but production is significantly increased under Pi limitation in a PhoB-dependent manner. Proteomic and lipidomic analyses revealed selective packaging of PhoB-regulated proteins involved in phosphate metabolism, stress response, carbon metabolism, and toxicity, as well as enrichment in phosphorus-free ornithine lipids under low Pi. Functional assays in Galleria mellonella demonstrated that OMVs from N16961 under Pi limitation are highly toxic, whereas OMVs from high-Pi or {Delta}phoB cultures exhibit minimal lethality. Our findings indicate that phosphate limitation acts as an environmental cue that shapes OMV composition, enhancing both bacterial survival and pathogenic potential. This study highlights OMVs as dynamic vehicles integrating adaptation to low Pi concentration, stress adaptation, and toxicity in V. cholerae.