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

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.

In Vitro Characterization of a Recombinant AHL-Lactonase from Bacillus cereus Isolated from a Striped Catfish (Pangasianodon hypophthalmus) Pond

“…AiiA N26.2 protein can be stored at 4°C for up to 5 days or at 20°C for up to 3 days, maintaining its activity at least 80 % of the maximal level. Our results are consistent with those reported by other authors [12,18,19]. These properties of AiiA N26.2 protein can facilitate its future oral administration via supplementation into fish/shrimp feed ingredients for efficient control of QS-regulated aquaculture pathogens.…”

“…Genetically-modified Erwinia carotovora and Pseudomonas aeruginosa expressing AHL-lactonase showed decreased production of virulence factors and attenuated virulence [15,16]. AHL-lactonase has been over-expressed successfully in E. coli and Pichia pastoris expression system as a soluble heterologous protein [17,18]. The purified recombinant AHL-lactonase originating from Bacillus sp.…”

“…The purified recombinant AHL-lactonase originating from Bacillus sp. strain B546 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 [18]. Recombinant AHL-lactonases, which allowed the blocking of the bacterial QS by hydrolyzing the AHL molecules, can be used as potential drugs to treat QS-regulated diseases or as supplement to antibiotic based treatments.…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Bacterially mediated quorum quenching using AiiA was also demonstrated to be beneficial against plant pathogens, both when aiiA was expressed in the pathogenic bacterium itself and during coinoculation of the pathogen with bacteria naturally producing AiiA or engineered bacterial strains expressing heterologous AiiA (88,89,(93)(94)(95)(96). AiiA was also shown to have potential for use in aquaculture, as carp fed either recombinant AiiA protein or Bacillus expressing aiiA were more resistant to infection by the fish pathogen Aeromonas hyrophila (361,362). Separately, AHLdegrading enrichment cultures (EC) of undefined microbial isolates naturally associated with marine organisms have been evaluated for use as probiotic cultures in aquaculture.…”

Isolation and Identification of a Novel Inducible Antibacterial Peptide from the Skin Mucus of Japanese Eel, Anguilla japonica

“…To control this disease of eel, 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 [9]. In this study, we compared antibacterial activities of acetone extracts and acidic extracts of five tissues of Japanese eel and chose the skin mucus as the source for further purification of antibacterial peptides.…”