| Summary: | <p>Abstract</p> <p>Background</p> <p><it>Aeromonas hydrophila </it>is a serious pathogen and can cause hemorrhagic septicemia in fish. To control this disease, antibiotics and chemicals are widely used which can consequently result in "superbugs" and chemical accumulation in the food chain. Though vaccine against <it>A. hydrophila </it>is available, its use is limited due to multiple serotypes of this pathogen and problems of safety and efficacy. Another problem with vaccination is the ability to apply it to small fish especially in high numbers. In this study, we tried a new way to attenuate the <it>A. hydrophila </it>infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in <it>Pichia pastoris</it>.</p> <p>Results</p> <p>The AHL-lactonase (AiiA<sub>B546</sub>) from <it>Bacillus </it>sp. B546 was produced extracellularly in <it>P. pastoris </it>with a yield of 3,558.4 ± 81.3 U/mL in a 3.7-L fermenter when using 3-oxo-C8-HSL as the substrate. After purification with a HiTrap Q Sepharose column, the recombinant homogenous protein showed a band of 33.6 kDa on SDS-PAGE, higher than the calculated molecular mass (28.14 kDa). Deglycosylation of AiiA<sub>B546 </sub>with Endo H confirmed the occurrence of <it>N</it>-glycosylation. The purified recombinant AiiA<sub>B546 </sub>showed optimal activity at pH 8.0 and 20°C, exhibited excellent stability at pH 8.0-12.0 and thermal stability at 70°C, was firstly confirmed to be significantly protease-resistant, and had wide substrate specificity. In application test, when co-injected with A. <it>hydrophila </it>in common carp, recombinant AiiA<sub>B546 </sub>decreased the mortality rate and delayed the mortality time of fish.</p> <p>Conclusions</p> <p>Our results not only indicate the possibility of mass-production of AHL-lactonase at low cost, but also open up a promising foreground of application of AHL-lactonase in fish to control <it>A. hydrophila </it>disease by regulating its virulence. To our knowledge, this is the first report on heterologous expression of AHL-lactonase in <it>P. pastoris </it>and attenuating <it>A. hydrophila </it>virulence by co-injection with AHL-lactonase.</p>
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