Identification And Characterization Of Intracellular Survival Genes Of Aeromonas Hydrophila

Intracellular survival is an important virulence factor of bacteria. Some pathogenic bacteria can evade the killing effects of phagocytes and remain activity or even growth and reproduction in phagocytes, which result in toxic effects and disease outbreaks finally. Pathogenic Aeromonas hydrophila bacteria isolated from diseased eels was used as the research object and its external infectious model were establish firstly;mutant library of A.hydrophila was constructed by transposon tagging technology, the defective intracellular survival mutants were screened from the mutant library and flanking mutant gene of transposon insertion were selected by genomic walking technology; Finally, by comparing growth, motility and cell adhesion and other biological characteristics of the mutant strain with wild-type strain, attempting to explore the mutated gene function in intracellular survival process of A.hydrophila. The results are as follows:First, the external infectious eel phagocytes quantitative model of A.hydrophila was established successfully. The model showed that the best infectious time of pathogenic A.hydrophila to peripheral blood of eel is 30 min and the best multiplicity of infection(bacterium: phagocytes) is 100:1.The model based on phagocytic rate and sterilization rate suggested that the phagocytic rate of A.hydrophila was higher than E. coli by the eel peripheral blood leukocyte, but the killing rate of A.hydrophila was much lower than Escherichia coli and Staphylococcus aureus, thus proved theis model had a good feasibility and lay a solid foundation for the future study on intracellular survival mechanism of A.hydrophila.Second, A.hydrophila mutant library was constructed by transposon(mini-Tn10) tagging technology, and three mutants with intracellular survival reduced significantly, AH109, AH159 and AH160 were screened. PCR identification further proved that mutation was caused by transposon insertion.Third, the flanking unknown sequences of transposon insertion sites were amplified by the Genome Walking PCR. After sequencing, the obtained sequences were blasted on line, and sequence homology analysis was showed that AH 109 mutant sequences was with the highest homology(97%) to hydrogenase-4 component G of A.hydrophila subsp. hydrophila ATCC 7966. AH 159 mutant sequences was with 95% homoloty to acetolactate synthase, large subunit, biosynthetic type ilvBEDA operon leader peptide of A.hydrophila subsp. hydrophila ATCC 7966. As for AH160, the mutant sequences showed 95% homology to AsmA family of A.hydrophila subsp. hydrophila ATCC 7966.Fourth, the biological characteristics of the mutant strain and wild strain, like growth, movement and adhesion, were studied in this research. The results suggested that not only the growth ability of AH109 and AH159 and the movement ability of AH160, but also the adhesion of three mutants were decreased significantly while compared with the wild train.In conclusion, gene hydrogenase, acetolactate synthase, and AsmA family may have impact on intracellular survival of A.hydrophila by growth, movement and adhesion et al. The study results lay the research foundation for study on pathogenic mechanism of A.hydrophila and provides a theoretical basis for the prevention and treatment of Aeromonas hydrophila disease.