Identification and characterization of the surface layer protein AvsA in outer membrane vesicles, antibiotic resistance, and in vivo host colonization in Aeromonas veronii

Outer membrane vesicles (OMVs) are important in bacterial communication and the transfer of virulence factors. In this study, we identified and characterized the surface layer protein (SLP) AvsA (Aeromonas veronii surface protein A) in the OMVs of Aeromonas veronii Hm21, a strain isolated from the medicinal leech Hirudo verbana. The surface layer proteins (SLPs) play critical roles in how bacteria interact with each other and their environments, particularly in mediating antibiotic resistance and facilitating host colonization. Furthermore, we investigate the ability of AvsA to confer protection against antibiotics, affect biofilm formation, and contribute to host colonization, providing insight into antibiotic resistance and two crucial factors contributing to the persistence of the bacteria in its host. Our findings suggest that AvsA enhances antibiotic tolerance, facilitates biofilm development, and is important for successful colonization of the leech digestive tract. These data demonstrate that AvsA performs important roles in a wide range of critical phenotypes. This work provides insights into the functional significance of SLPs in A. veronii and highlights AvsA as a potential target for modulating bacterial colonization and resilience against antibiotics. ImportanceOuter membrane vesicles (OMVs) are important for bacterial communication, pathogenesis, and stress adaptation, yet how this is accomplished remains poorly understood. Here, we identify and characterize a surface layer protein (SLP), AvsA, associated with OMVs in Aeromonas veronii Hm21. Bioinformatic, phylogenetic, and mass spectrometry analyses suggest that Aeromonas veronii ORF M001_06550 encodes a surface layer protein (SLP) with high similarity to a characterized A. hydrophila SLP, supporting its designation as Aeromonas veronii Surface Protein A (AvsA). AvsA forms a paracrystalline layer on bacterial cells and OMVs. Functionally, AvsA contributes to antibiotic resistance, enhances biofilm formation, and is essential for colonization in a symbiotic host, the medicinal leech. These findings highlight a novel role of SLPs in bacterial physiology and host interactions. Given the widespread presence of AvsA homologs, our study provides insights into conserved bacterial mechanisms that may be relevant for both pathogenic and beneficial host-microbe interactions.