Characterization of proteins present in the biofilm matrix and outer membrane vesicles of during iron-sufficient and iron-restricted growth: identification of potential protective antigens through analyses.

There is limited efficacy in vaccines currently available to prevent some animal diseases of bacterial origin, such as bovine respiratory disease caused by . Protective efficacy can potentially be improved if bacterial antigens that are expressed in the host are included in vaccines. During infection in the bovine host, biofilms become established, and the availability of essential iron is restricted. To investigate further, the protein composition of spontaneously released outer membrane vesicles (OMVs) during iron-sufficient and iron-restricted growth and the proteins expressed in the biofilm matrix were analyzed and compared. Proteomic analysis revealed a dramatic physiological change in as it transitioned from the planktonic form to the biofilm mode of growth. All transferrin-binding proteins (Tbps) previously identified in were detected in the OMVs, suggesting that OMVs could induce antibodies to these proteins. Two TbpA-like proteins and seven total proteins were present in the OMVs only when iron was restricted, indicating the expression of these Tbps was differentially regulated. More proteins associated with quorum-sensing (QS) signaling were detected in the biofilm matrix compared with proteins in the OMVs, supporting a link between QS and biofilm formation. Proteins ACA31267.1 (OmpA) and ACA32419.1 (TonB-dependent receptor) were present in the OMV and biofilm matrix and predicted to be potential protective antigens using an immuno-bioinformatic approach. Overall, the results support the development of novel vaccines that contain OMVs obtained from bacteria grown to simulate the environment, and possibly biofilm matrix, to prevent diseases caused by bacterial pathogens.IMPORTANCEBovine respiratory disease (BRD) is the most economically important disease affecting the cattle industry. Available BRD vaccines consist of killed bacteria but are not very effective. Poor vaccine efficacy may be because the phenotype of bacteria in the host differs from the phenotype of cultured bacteria. Following host infection, virulent bacteria can express transferrin-binding proteins (Tbps) not expressed in culture medium but are required to sequester iron from host proteins. During chronic infections, such as BRD, bacteria can form a biofilm consisting of novel protein and polysaccharide antigens. The unique proteins expressed on outer membrane vesicles (OMVs) of (a BRD pathogen) in the absence of iron and as a biofilm were identified and characterized. At least two TbpA-like proteins were expressed in OMVs only under iron-limiting conditions. Quorum-sensing-associated proteins were identified in the biofilm matrix. analysis identified potential protein targets for vaccine development.