Heterologous expression of AHL lactonase AiiK by  Lactobacillus casei  MCJΔ1 with great quorum quenching ability against  Aeromonas hydrophila  AH-1 and AH-4

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Abstract

Background

Nowadays, microbial infections have caused increasing economic losses in aquaculture industry and deteriorated worldwide environments. Many of these infections are caused by opportunistic pathogens through cell-density mediated quorum sensing (QS). The disruption of QS, known as quorum quenching (QQ), is an effective and promising way to prevent and control pathogens, driving it be the potential bio-control agents. In our previous studies, AHL lactonase AiiK was identified with many characteristics, and constitutive expression vector pELX1 was constructed to express heterologous proteins in Lactobacillus casei MCJΔ1 ( L. casei MCJΔ1). In this study, recombinant strain pELCW- aiiK/ L. casei MCJΔ1 ( LcAiiK) and wild-type Aeromonas hydrophila ( A. hydrophila) were co-cultured to test the QQ ability of LcAiiK against A. hydrophila.

Results

A cell wall-associated expression vector pELCW for L. casei MCJΔ1 was constructed. Localization assays revealed that the expressed AiiK was anchored at the surface layer of LcAiiK via vector pELCW- aiiK. LcAiiK (OD ₆₀₀ = 0.5) degraded 24.13 μM of C ₆-HSL at 2 h, 40.99 μM of C ₆-HSL at 12 h, and 46.63 μM of C ₆-HSL at 24 h. Over 50% LcAiiK cells maintained the pELCW- aiiK plasmid after 15 generations of cultivation without erythromycin. Furthermore, LcAiiK inhibited the swimming motility, extracellular proteolytic activity, haemolytic activity and biofilm formation of A. hydrophila AH-1 and AH-4.

Conclusion

The AHL lactonase AiiK is firstly and constitutively expressed at the surface layer of L. casei MCJΔ1. LcAiiK displayed considerable AHL lactonase activity and great QQ abilities against A. hydrophila AH-1 and AH-4 by attenuating their QS processes instead of killing them. Therefore, the LcAiiK can be exploited as an anti-pathogenic drug or a bio-control agent to control the AHL-mediated QS of pathogenic bacteria.

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Most cited references 41

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Gene splicing and mutagenesis by PCR-driven overlap extension.

Karin L Heckman, Larry R Pease (2007)

Extension of overlapping gene segments by PCR is a simple, versatile technique for site-directed mutagenesis and gene splicing. Initial PCRs generate overlapping gene segments that are then used as template DNA for another PCR to create a full-length product. Internal primers generate overlapping, complementary 3' ends on the intermediate segments and introduce nucleotide substitutions, insertions or deletions for site-directed mutagenesis, or for gene splicing, encode the nucleotides found at the junction of adjoining gene segments. Overlapping strands of these intermediate products hybridize at this 3' region in a subsequent PCR and are extended to generate the full-length product amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. The highly efficient generation of mutant or chimeric genes by this method can easily be accomplished with standard laboratory reagents in approximately 1 week.

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The evolution of cooperation within the gut microbiota

Seth Rakoff-Nahoum, Kevin R. Foster, Laurie E. Comstock (2016)

Cooperative phenotypes are considered central to the functioning of microbial communities in many contexts, including communication via quorum sensing, biofilm formation, antibiotic resistance, and pathogenesis 1-5 . The human intestine houses a dense and diverse microbial community critical to health 1,2,4-9 , yet we know little about cooperation within this important ecosystem. Here we experimentally test for evolved cooperation within the Bacteroidales, the dominant Gram-negative bacteria of the human intestine. We show that during growth on certain dietary polysaccharides, the model member Bacteroides thetaiotaomicron exhibits only limited cooperation. Although this organism digests these polysaccharides extracellularly, mutants lacking this ability are outcompeted. In contrast, we discovered a dedicated cross-feeding enzyme system in the prominent gut symbiont Bacteroides ovatus, which digests polysaccharide at a cost to itself but at a benefit to another species. Using in vitro systems and gnotobiotic mouse colonization models, we find that extracellular digestion of inulin increases the fitness of B.ovatus due to reciprocal benefits when it feeds other gut species such as Bacteroides vulgatus. This is a rare example of naturally-evolved cooperation between microbial species. Our study reveals both the complexity and importance of cooperative phenotypes within the mammalian intestinal microbiota.

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Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent.

Michael Givskov, Mette Burmølle, Kit Madsen … (2005)

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell-cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2, and are readily phagocytosed by PMNs, in contrast to bacteria with functional QS systems. In contrast to the wild-type, QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently, the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

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Author and article information

Contributors

Shumiao Zhao:

ORCID: http://orcid.org/0000-0001-6047-8880

shumiaozhao@mail.hzau.edu.cn

Journal

Journal ID (nlm-ta): Microb Cell Fact

Journal ID (iso-abbrev): Microb Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2859

Publication date (Electronic): 7 October 2020

Publication date PMC-release: 7 October 2020

Publication date Collection: 2020

Volume: 19

Electronic Location Identifier: 191

Affiliations

[1 ]GRID grid.35155.37, ISNI 0000 0004 1790 4137, State Key Laboratory of Agricultural Microbiology and College of Life Science and Technology, , Huazhong Agricultural University, ; Wuhan, 430070 China

[2 ]GRID grid.464330.6, Institute of Agricultural Resources and Regional Planning, CAAS, ; Beijing, 100081 China

Author information

Shumiao Zhao http://orcid.org/0000-0001-6047-8880

Article

Publisher ID: 1448

DOI: 10.1186/s12934-020-01448-4

PMC ID: 7542731

PubMed ID: 33028330

SO-VID: 4e8fc698-440c-44af-aa59-c70ad86004c9

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 21 November 2019

Date accepted : 25 September 2020

Funding

Funded by: Fundamental Research Funds for Central Universities

Award ID: 2662018JC016

Award Recipient : Shumiao Zhao

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: quorum sensing,quorum quenching,ahl lactonase aiik,lactobacillus casei mcjδ1,aeromonas hydrophila

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ScienceOpen disciplines: Biotechnology

Keywords: quorum sensing, quorum quenching, ahl lactonase aiik, lactobacillus casei mcjδ1, aeromonas hydrophila

Heterologous expression of AHL lactonase AiiK by Lactobacillus casei MCJΔ1 with great quorum quenching ability against Aeromonas hydrophila AH-1 and AH-4

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Genome Engineering using CRISPR

Most cited references 41

Gene splicing and mutagenesis by PCR-driven overlap extension.

The evolution of cooperation within the gut microbiota

Pseudomonas aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum-sensing dependent.

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