Cloning and Expression of Listeriolysin O in Lactobacillus plantarum

Abstract

Background: The protein listeriolysin O (LLO) encoded by hly gene, is one of the most important virulence factors of Listeria monocytogenes, responsible for phagosomal membrane disruption and bacterial escape to the cytoplasm, stimulation of CD8+ T cells and Th1 response. Recently pathobiotechnological vaccination using probiotic bacteria have been proposed. One of these strategies is expression of LLO in non-pathogenic bacteria such as lactic acid bacteria as delivery strains. Objective: In the current study, we aimed to clone hly gene in a Lactobacillus species via pNZ8110, an inducible expression vector which is specific for Lactococcus species. Materials and Methods: Hly gene was amplified by polymerase chain reaction (PCR) and inserted into pNZ8110 by restriction enzymes cutting and ligation method. After transformation and propagation in Escherichia coli MC1061 intermediate host, it was successfully electrotransformed into Lactobacillus plantarum. Results: Gel electrophoresis of colony PCR, extracted plasmids and restriction analysis along with sequencing confirmed the transformation. After induction with supernatant of nisin producer, strain Lactococcus lactis NZ9700, expression of LLO was confirmed by SDS-PAGE and western blot. Conclusion: Here, we employed a nonpathogenic probiotic strain, L. plantarum for the first time to express hly gene of L. monocytogenes in order to propose a new vaccine candidate.