Outer Membrane Vesicles from bacteria expressing the HlyF/CprA family of enzymes are more efficient at delivering their cargo into host cells.

HlyF, a cytoplasmic short-chain dehydrogenase/reductase encoded by ColV-like virulence plasmids in Escherichia coli, and its ortholog CprA, encoded on the chromosome of Pseudomonas aeruginosa, drive the production of a distinct population of bacterial outer membrane vesicles (OMVs). These vesicles were previously shown to disrupt autophagic flux and amplify host inflammatory responses, underscoring their functional divergence from canonical OMVs. Here, we demonstrate that HlyF/CprA-dependent OMVs also display enhanced cargo delivery efficiency through a dynamin-independent internalization pathway involving direct fusion with the host cell plasma membrane. This non-canonical uptake mechanism differentiates these vesicles from conventional bacterial OMVs and promotes efficient cytosolic delivery of vesicular cargo. Collectively, our findings reveal a previously unrecognized role for the HlyF/CprA enzyme family in modulating OMVs biogenesis and functional properties, and identify a novel OMV-mediated mechanism contributing to bacterial virulence.