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 pathogenic Pseudomonas fluorescens strain, TSS, isolated from diseased fish. The expression of pfa1 is enhanced during infection and is regulated by growth phase and growth conditions. Mutation of pfa1 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 by pfa1 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 from Escherichia 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 degrees 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 in E. coli, AT1 was able to deliver an integrated Edwardsiella tarda immunogen (Et18) onto the surface of bacterial cells. Compared to purified recombinant Et18, Et18 displayed by E. coli via AT1 induced significantly enhanced immunoprotection.
Hu
Yong-hua
YH
Institute of Oceanology, Chinese Academy of Sciences, Qingdao, People's Republic of China.
Liu
Chun-sheng
CS
Hou
Jin-hui
JH
Sun
Li
L
D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism; D001428:Bacterial Vaccines / Q000235:genetics / Q000276:immunology; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D020609:Edwardsiella tarda / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000382:microbiology; D017353:Gene Deletion; D015870:Gene Expression; D020869:Gene Expression Profiling; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011552:Pseudomonas Infections / Q000382:microbiology / Q000662:veterinary; D011551:Pseudomonas fluorescens / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013696:Temperature; D014774:Virulence; D037521:Virulence Factors / Q000096:biosynthesis / Q000235:genetics*
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D002352:Carrier Proteins; D037521:Virulence Factors; D010447:Peptide Hydrolases