sRNA-enriched outer membrane vesicles of Vibrio alginolyticus: Decisive architects of biofilm assembly.

Biofilms robustly defend bacteria by impeding antimicrobial entry, and protecting against environmental stress and immune responses, thereby enhancing host colonization. Overcoming the diagnostic and therapeutic challenges of biofilm infections is critical, given their clinical significance and resistance to standard treatments. Recent investigations have illuminated the pivotal role of outer membrane vesicles (OMVs) in biofilm formation. This study delves into the OMVs secreted by Vibrio alginolyticus, a zoonotic species and the second largest class of pathogenic Vibrio. We successfully extracted, purified, and characterized OMVs from V. alginolyticus. Our data reveal that these OMVs not only facilitate biofilm formation but are also enriched with small RNAs (sRNAs). Bioinformatics analyses suggest that these sRNAs, characterized by distinct secondary structures and targeting various regulatory genes, may be integral to the biofilm formation process. To verify this, we constructed sRNA-deficient mutants and OMVs, then evaluated their effects on bacterial physiology, including growth, motility, extracellular matrix production, and biofilm development. Our findings demonstrate that sRNA cargos play a crucial role in these processes and modulate gene expression associated with biofilm formation. This study highlights the significant role of OMVs delivering sRNAs in regulating bacterial biofilm formation, offering new insights into bacterial pathogenesis and resistance mechanisms.