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.

Construction of a recombinant Lactobacillus casei expressing fliC gene fused with guanylyl cyclase C and dendritic cell-binding peptide using CRISPR–Cas9 system: a first step towards design of vaccine against colorectal cancer

“…In a study conducted by One more limitation is lower transformation efficiency in lactobacilli compared with some other bacteria, making it essential for the electroporation protocol to be optimized for each species, even within the strains of one species [31]. In a study conducted by Hayati et al, several Lactobacillus species including L. casei, Lactobacillus gasseri, Lactobacillus acidophilus and L. plantarum were used for the cloning of Listeria monocytogenes listeriolysin O by using inducible expression plasmid, pNZ8110, that only transformation in L. plantarum was happened [32]. Fortunately, the transformation was successfully performed in our study, although there was a limited number of transformants.…”