Identification, Characterization, and Molecular Application of a Virulence-Associated Autotransporter from a Pathogenic Pseudomonas fluorescens Strain

A gene,pfa1 , encoding an autotransporter was cloned from a pathogenicPseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression ofpfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation ofpfa1 significantly attenuates the overall bacterial virulence of TSS and impairs the abilities of TSS in biofilm production, interaction with host cells, modulation of host immune responses, and dissemination in host blood. The putative protein encoded bypfa1 is 1,242 amino acids in length and characterized by the presence of three functional domains that are typical for autotransporters. The passenger domain of PfaI contains a putative serine protease (Pap) that exhibits apparent proteolytic activity when expressed in and purified fromEscherichia coli as a recombinant protein. Consistent with the important role played by PfaI in bacterial virulence, purified recombinant Pap has a profound cytotoxic effect on cultured fish cells. Enzymatic analysis showed that recombinant Pap is relatively heat stable and has an optimal temperature and pH of 50°C and pH 8.0. The domains of PfaI that are essential to autotransporting activity were localized, and on the basis of this, a PfaI-based autodisplay system (named AT1) was engineered to facilitate the insertion and transport of heterologous proteins. When expressed inE. coli , AT1 was able to deliver an integratedEdwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed byE. coli via AT1 induced significantly enhanced immunoprotection.