High yield expression of an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture

Ruidong Chen,Yanan Cao,Yingguo Bai,Zhigang Zhou,Bin Yao

doi:10.1186/1475-2859-9-39

Ruidong Chen, Yanan Cao + Show 3 more

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https://doi.org/10.1186/1475-2859-9-39

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Abstract

BackgroundAeromonas hydrophila 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 A. hydrophila 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 A. hydrophila infection by using a quorum quenching strategy with a recombinant AHL-lactonase expressed in Pichia pastoris.ResultsThe AHL-lactonase (AiiAB546) from Bacillus sp. B546 was produced extracellularly in P. pastoris 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 AiiAB546 with Endo H confirmed the occurrence of N-glycosylation. The purified recombinant AiiAB546 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. hydrophila in common carp, recombinant AiiAB546 decreased the mortality rate and delayed the mortality time of fish.ConclusionsOur 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 A. hydrophila disease by regulating its virulence. To our knowledge, this is the first report on heterologous expression of AHL-lactonase in P. pastoris and attenuating A. hydrophila virulence by co-injection with AHL-lactonase.

Highlights

Aeromonas hydrophila is a serious pathogen and can cause hemorrhagic septicemia in fish
Gene cloning and sequence analysis Using the primers BT1 and BT2 designed specific for N-acyl homoserine lactones (AHLs)-lactonases of Bacillus spp., the full-length 753-bp AHL-lactonase gene, aiiAB546, was cloned from Bacillus sp
Expression and fermentation of recombinant AiiAB546 in P. pastoris The AHL-lactonase gene aiiAB546 was transformed into P. pastoris GS115 competent cells with pPIC9 vector

Summary

Introduction

Aeromonas hydrophila is a serious pathogen and can cause hemorrhagic septicemia in fish. Aeromonas hydrophila is a Gram-negative rod and behaves as an opportunistic pathogen in both aquatic and host environments [1,2,3] It can cause hemorrhagic septicemia, resulting in fin and tail rot and epizootic ulcerative syndrome in juvenile and mature fish or intestinal and wound infection in humans [3,4,5,6,7]. That A. hydrophila harbors the AhyI/AhyR quorum-sensing system, utilizes AHL-dependent quorum-sensing to regulate the expression of virulent genes, and mediates the process of microbial infection and colonization in the host provide a potentially promising strategy to control A. hydrophila--quorum-quenching [1,6,7,10,13]. Unlike AHL-acylase and PONs, which have variable substrate spectra, AHL-lactonase shows distinct substrate specificity and only efficiently hydrolyses AHL signals [20,21,24]

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