Screening Of OMVs-transported Bacterial Virulence Factors In Edwardsiella Piscicida And Preliminary Investigation Of Their Roles During Infection

Bacterial outer membrane vesicles(OMVs)are lipid bilayer spherical vesicles secreted by Gram-negative bacteria,which carry a variety of bacterial virulence factors and transport them either to the bacterial extracellular environment or further into the host cells.Therefore,OMVs are considered as an important way for bacteria to transport virulence factors.Edwardsiella piscicida is an important fish pathogen,which can transport bacterial virulence factors into the host cells via T3SS and T6SS,and thereby promoting the bacteria to establish intracellular infection.In this work,we explored whether the outer membrane vesicles were involved in the transport of bacterial virulence factors in E.piscicida and further investigated the roles of bacterial hemolysin on their internalization,intracellular vesicle trafficking and immune recognition.This work includes the following aspects:First,the extraction process of OMVs was established by ultrafiltration concentration and density gradient centrifugation.We observed the morphology of OMVs by electron microscopy.The time phase changes of OMVs produced during the growth of E.piscicida were investigated,and the protein profile of OMVs was preliminarily analyzed by SDS-PAGE.Next,we analyzed protein components of OMVs by mass spectrometry and identified many virulence factors,including T3SS components,T6SS components,bacterial iron uptake system components and hemolysin.In addition,we used immunoblotting to screen for OMVs-transported bacterial effectors from the intracellularly transported bacterial proteins.Finally,we examined the basic features of OMVs entering host cells and intracellular vesicle trafficking,and investigated the roles of hemolysin on the internalization,intracellular vesicle trafficking and immune recognition of E.piscicida OMVs.Our data suggested that hemolysin cannot affect the internalization of OMVs,but it can promote OMVs to escape from the early endocytic vesicles into the cytoplasm,which in turn promoted the immune recognition of OMV-contained LPS by the cytosolic sensor,caspase 4.In summary,this work identified a variety of OMV-transported bacterial virulence factors in E.piscicida,and further explored the effects of hemolysin on intracellular processes of E.piscicida OMVs,which lays a foundation for further interpreting the pathogenesis of E.piscicida.