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Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000.

ABSTRACT: BACKGROUND: Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under inducible conditions using the NICE (NIsin Controlled Expression) system. We created a modified pNZ8048 vector encoding a six-His-tagged LLO downstream of the strong inducible PnisA promoter. RESULTS: The constructed vector (pNZPnisA:CYTO-LLO) was expressed in L. lactis NZ9000 and was best induced at mid-log phase with 0.2% v/v nisin for 4 h statically at 30 degrees C. Purification of the His-tagged LLO was accomplished by Ni-NTA affinity chromatography and functionality was confirmed through haemolytic assays. Total LLO yield (measured as total protein content) was 4.43-5.9 mg per litre culture and the haemolytic activity was still detectable after 8 months of storage at 4 degrees C. CONCLUSION: The LLO production method described in this work provides an approach to efficient LLO production in the Gram-positive Lactococcus bacterium to yield a significant source of the protein for research and diagnostic applications. Expression of LLO in L. lactis has a number of benefits over E. coli which may facilitate both in vivo and in vitro applications of this system.

SUBMITTER: Bahey-El-Din M

PROVIDER: S-EPMC2515284 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000.

Bahey-El-Din Mohammed M Griffin Brendan T BT Gahan Cormac Gm CG

Microbial cell factories 20080729

<h4>Background</h4>Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals. Listeriolysin O (LLO) is the major virulence factor of the pathogen and is often used as a diagnostic marker for detection of L. monocytogenes. In addition, LLO represents a potent antigen driving T cell-mediated immunity during infection. In the present work, Lactococcus lactis NZ9000 was used as an expression host to hyper-produce LLO under induci ...[more]

PMID: 18664263

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Project description:Circular bacteriocins, also known as bacterial head-to-tail cyclized peptides, are a subgroup of ribosomally synthesized and post-translationally modified peptides (RiPPs). Compared with their conventional linear counterparts, circular bacteriocins are highly stable over a broad temperature and pH range, and circularization decreases proteolytic degradation by exopeptidases. These features render them great potential as scaffold candidates to withstand strident conditions in food- and pharmaceutical applications. However, the biosynthesis and bioactivity of circular bacteriocins still remain largely unknown. To investigate and gain more insights into the biosynthesis of circular bacteriocins and to achieve efficient production and characterization of bacteriocin variants, we developed an efficient cloning and heterologous expression system for clostridial circularin A and successfully produced this circular peptide in Lactococcus lactis NZ9000. We report three system formats with single plasmid or plasmid combinations to achieve successful cloning and functional production of circularin A in L. lactis. These systematic varieties enabled us to choose the appropriate method to efficiently obtain various constructs with desired properties. With the established heterologous systems in L. lactis, we performed several mutagenesis studies in the precursor peptide to study its structure/function relationships. The overlay activity assay revealed that these mutant variants had variable effects on different indicator strains: lysine substitution for certain glutamine residue(s) greatly decreased its bioactivity against Clostridium perfringens and L. lactis NZ9000, and alanine replacement for the cationic residues significantly reduced the activity against Lactobacillus sake ATCC 15521, whereas alanine substitution for the aromatic residues decreased its bioactivity against all three testing strains dramatically. Moreover, the conditions for bacteriocin production were optimized. Results show that supplementing the minimal medium with extra glucose (or sucrose) and immediate nisin-induction improved the peptide yield significantly. Briefly, we developed an excellent system for the production of circularin A and a wide range of variant peptides in a convenient host, as well as a method for fast detection of peptide production and activity. This system facilitated our mutagenesis studies which provided valuable insights into the effects of mutating specific residues on its biosynthesis and bioactivity, and will eventually enable more complex research into the biosynthesis of circularin A.

Dataset Information

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000.

Publications

Nisin inducible production of listeriolysin O in Lactococcus lactis NZ9000.

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