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

Abstract

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, Lbc^{Inl AB} , 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 Lbc^{Inl AB} was confirmed by Western blotting and immunofluorescence staining. The Lbc^{Inl AB} strain showed significantly higher (P < 0.0001) adherence, invasion and translocation of Caco-2 cells than the wild-type L. casei strain (Lbc^WT ), as well as reduced L. monocytogenes adhesion, invasion and transcellular passage through the cell monolayer than Lbc^WT . Furthermore, pre-exposure of Caco-2 cells to Lbc^{Inl AB} 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.

Figure 1

Agarose gel showing (A) PCR‐amplified gene products for inlAB, inlA and inlB of InlAB‐expressing 3 recombinant Lactobacillus casei strains (Lbc^{Inl AB −1}, Lbc^{Inl AB −2}, Lbc^{Inl AB −3}) and L. monocytogenes (Lm) and Lbc^WT. Lm: L. monocytogenes F4244 (positive control) and Lbc^WT (negative control).B. Western blot showing expression of Internalins InlA and InlB in the recombinant L. casei strains (Lbc^{Inl AB −1}, Lbc^{Inl AB −2}, Lbc^{Inl AB −3}, Lbc^WT and Lbc^V) in the different cellular fractions (supernatant, cell wall and intracellular) and L. monocytogenes F4244 (Lm). C. Immunofluorescence staining of bacteria (magnification 1000 × ) with anti‐InlA mAb‐2D12 and anti‐InlB pAb404. Lbc^{Inl AB} and Lm (control) cells indicated the presence of InlA (green) and no expression in Lbc^WT. Anti‐InlB pAb‐404 staining produced weak signal, suggesting this antibody may not be suitable for immunofluorescence staining.

Figure 2

Panel showing L. casei growth curves. (A) Optical density measurement (OD at 600 nm), (B) bacterial counts and (C) phase‐contrast microscopic images of Lbc^WT, Lbc^V and Lbc^{Inl AB}. This experiment was performed twice in triplicates.

Figure 3

Adhesion, invasion and translocation profiles of Listeria monocytogenes (Lm) and Lactobacillus casei (Lbc) to Caco‐2 cells.A. Adhesion, (B) invasion and (C) translocation of the Caco‐2 cells by L. monocytogenes and L. casei strains (Lbc^WT, Lbc^V, Lbc^{Inl AB} and Lbc^LAP). Percentages were calculated relative to the inoculums that were added to the Caco‐2 cells. Data are average (SD) of three independent experiments performed in duplicate. For each time point, bars marked with different letters (a, b, c, d) indicate significant difference at P < 0.05.

Figure 4

Competitive exclusion of Listeria monocytogenes (Lm) adhesion to Caco‐2 cells by L. casei strains (Lbc^WT, Lbc^V, Lbc^{Inl AB} and Lbc^LAP), analysed by three different exclusion mechanisms: (A) competitive adhesion – Caco‐2 cells were exposed to L. casei strains with Lm simultaneously; (B) inhibition of adhesion – Caco‐2 cells were pre‐exposed to L. casei strains for 1 h before infection with Lm; and (C) displacement of adhesion – Caco‐2 cells were infected with Lm for 1 h before L. casei treatment (1 h). Adhesion of Lm alone to Caco‐2 cells was presented as 100%, and per cent adhesion was calculated relative to that. For each time point, bars marked with different letters (a, b, c) indicate significant difference at P < 0.05.

Figure 5

Inhibition of Listeria monocytogenes (Lm) adhesion (A), invasion (B) and transcellular translocation (C) by the L. casei strains (Lbc^WT, Lbc^V, Lbc^{Inl AB} and Lbc^LAP). Caco‐2 cells were pre‐exposed to L. casei strains for 1, 4, 16 and 24 h before infection with Lm for 1 h for adhesion and invasion and 2 h for translocation. Data are averages of three experiments ran in duplicates. For each time point, bars marked with different letters (a, b, c, d, e, f, g, h) indicate significant difference at P < 0.05.

Figure 6

Cytotoxicity of Listeria monocytogenes in Caco‐2 cells pre‐exposed with Lactobacillus casei over time (1, 4, 16, 24 h). Cytotoxicity value for L. monocytogenes treatment (1 h) in the absence of L. casei strains was 64.38%. Data are averages of three experiments ran in duplicates. For each time point, bars marked with different letters (a, b, c, d, e, f, g) indicate significant difference at P < 0.05.

Figure 7

Caco‐2 cell permeability analysis using (A) transepithelial electrical resistance (TEER) and (B) 4 kDa of dextran^FITC (FD4) permeability assay. Caco‐2 cell monolayers were grown in trans‐well inserts and treated with L. casei strains (Lbc^WT, Lbc^V, Lbc^{Inl AB} or Lbc^LAP) for 2, 4, 16 and 24 h, before their infection with L. monocytogenes (Lm) for 2 h. TEER measurements before and after exposure to L. monocytogenes treatment alone were 268.9 ± 2.3 and 224.5 ± 4.7, respectively, with a 16.5% change. Values are averages of two experiments analysed in triplicate. Per cent TEER reduction was calculated as per Koo et al. (Koo et al., 2012) as 1 – TEER _after/TEER _before ×100.B. FD4 recovery after Lm was 2.76 ± 0.03%. Values are averages of three independent experiments performed in triplicates. For each time point, bars marked with different letters (a, b, c, d) indicate significant difference at P < 0.05.