Lactobacillus casei expressing Internalins A and B reduces Listeria monocytogenes interaction with Caco‐2 cells in vitro

Abstract

Summary Listeria monocytogenes has been implicated in a number of outbreaks including the recent largest outbreak in South Africa. Current methods for prevention of foodborne L. monocytogenes infection are inadequate, thus raising a need for an alternative strategy. Probiotic bioengineering is considered a prevailing approach to enhance the efficacy of probiotics for targeted control of pathogens. Here, the ability of Lactobacillus casei expressing the L. monocytogenes invasion proteins Internalins A and B (inlAB) to prevent infection was investigated. The inlAB operon was cloned and surface‐expressed on L. casei resulting in a recombinant strain, LbcInlAB, and subsequently, its ability to inhibit adhesion, invasion and translocation of L. monocytogenes through enterocyte‐like Caco‐2 cells was examined. Cell surface expression of InlAB on the LbcInlAB was confirmed by Western blotting and immunofluorescence staining. The LbcInlAB strain showed significantly higher (P < 0.0001) adherence, invasion and translocation of Caco‐2 cells than the wild‐type L. casei strain (LbcWT), as well as reduced L. monocytogenes adhesion, invasion and transcellular passage through the cell monolayer than LbcWT. Furthermore, pre‐exposure of Caco‐2 cells to LbcInlAB significantly reduced L. monocytogenes‐induced cell cytotoxicity and epithelial barrier dysfunction. These results suggest that InlAB‐expressing L. casei could be a potential practical approach for prevention of listeriosis.

Internalin AB-expressing recombinant Lactobacillus casei protects Caco-2 cells from Listeria monocytogenes-induced damages under simulated intestinal conditions

“…casei strains exhibited desired beneficial effects compared to the wild type strain. Furthermore, the recombinant strains did not exhibit any cytotoxic response in the in vitro experiments [21,26] including this study suggesting these recombinant strains possibly does not have negative effect. However, animal studies are needed to fully validate their health beneficial effects.…”

“…monocytogenes in L . casei (Lbc InlAB ) has been reported before [26] where the pLP401T vector [34] contains the origin of replication of Lactobacillus and the α-amylase promoter gene (Pamy). The promoter is mannitol inducible, therefore, modified MRS supplemented with 1% mannitol was used to induce the expression of InlAB in L .…”

“…casei and transcription terminator of the cbh (conjugated bile acid hydrolase) gene (Tcbh). The surface expression of InlAB on Lbc InlAB is envisaged to covalently link to the cell wall/peptidoglycan via anchor peptide and LPxTG motif [26]. L .…”

“…Due to promising results reported in these aforementioned studies, we also recently cloned and expressed the invasion operon inlAB into Lactobacillus casei (Lbc InlAB ) for the inhibition of L . monocytogenes adhesion, invasion and translocation in vitro , using the Caco-2 cells grown and maintained in the cell culture media, Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) [26]. The resultant recombinant probiotic showed enhanced inhibition of L .…”

Screening, Safety Evaluation, and Mechanism of Two Lactobacillus fermentum Strains in Reducing the Translocation of Staphylococcus aureus in the Caco-2 Monolayer Model

“…L. fermentum NCU3087 was found to display strong adaption to gastrointestinal fluids while L. fermentum NCU3088 was slightly weaker (Figure 5). Both L. fermentum NCU3087 and L. fermentum NCU3088 had a translocation rate of less than 0.01%, similar to the translocation rate of L. casei in a Caco-2 model (Mathipa et al, 2019). Susceptibility of L. fermentum NCU3087 and L. fermentum NCU3088 to 15 antibiotics were tested, the breakpoints used was in accordance with current European Food Safety Authority (EFSA) guidelines and the results corresponded to the antibiotic resistance among commercially available probiotics (Sharma et al, 2014).…”