Abstract
BackgroundListeria 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.ResultsThe 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°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°C.ConclusionThe 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.
Highlights
Listeria monocytogenes is a well-characterized food-borne pathogen that infects pregnant women and immunocompromised individuals
Listeria monocytogenes is the causative agent of listeriosis, a food-borne disease generally associated with the consumption of contaminated ready-to-eat food products
DNA sequence of the six His-tagged hly gene Native Listeriolysin O (LLO) secreted by Listeria monocytogenes EGD-e is composed of a secretory signal peptide (25 amino acids) and the actual active portion of LLO (504 amino acids)
Summary
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. LLO represents a potent antigen driving T cell-mediated immunity during infection. Listeria monocytogenes is the causative agent of listeriosis, a food-borne disease generally associated with the consumption of contaminated ready-to-eat food products. Listeriosis affects mainly immunocompromised individuals and the outcome of infection includes spontaneous miscarriage in pregnant women, and meningitis in the newborn [1]. Several common-source outbreaks of listeriosis have been linked to consumption of Listeria-contaminated foods (frankfurters, pâté, pasteurised milk and soft-cheese) [2]. Controlled good manufacturing procedures and early detection of Listeria contamination in food are of great importance in preventing such outbreaks
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