Next, we performed serial fractionation experiments in order to determine where the recombinant proteins are localized within the cell envelope of L. plantarum (Fig. 3A) . We treated recombinant L. plantarum with Lysozyme (Lyz) for 45 min and separated the cell wall fraction from the protoplast by centrifugation. We then disrupted the cells and separated the membrane from the cytosol fractions by ultracentifugation. The three fractions were then analyzed on denaturing polyacrylamide gels and OspA was identified by Immunoblot with anti-OspA monoclonal antibody (mAb LA2.2). Lipidated OspA expressed in L. plantarum is exported through the membrane and accumulates in the cell wall, while unlipidated OspA D17 remains mostly attached to the membrane (Fig. 3A) . Next, we wanted to analyze the localization of the recombinant proteins on the surface of L. plantarum. We incubated live recombinant L. plantarum with and without Lyz and we performed both live-cell ELISA (lcELISA) and immunofluorescence (IFA) assays. For lcELISA, we incubated the recombinant L. plantarum with Lyz for 5 and 45 min, the cells were washed and incubated with mAb LA2.2 (Fig. 3B) . For immunofluorescence, we performed a 30 min incubation with Lyz after which the cells were washed, incubated with mAb LA2.2 followed by Fluor 488-labeled goat anti-mouse IgG (1:250). Staining was visualized using a Zeiss inverted Axiovert 200 microscope (Fig. 3C) . In both assays, IFA and lcELISA, reactions without lyz (No Lyz) detect protein that is exposed on the surface of the cell. The epitope used to detect OspA and OspA D17 is located in the carboxyl terminal of the protein and is not surface exposed ( Fig. 3B and 3C) . Reactions with Lyz digest the peptidoglycan that releases the OspA that is trapped within the cell wall and expose OspA that is attached to the membrane (Lyz 5 min, 45 min or 30 min, Fig. 3B and 3C ). Our results indicate that lipidated OspA (LpA) is trapped within the cell wall. That is, a 5 min digestion with Lyz exposes a high amount of OspA that is associated with the peptidoglycan layer (Fig. 3B ) and a 45 min or 30 min digestion exposes a lower amount of OspA that is associated with the peptidoglycan layer ( Fig. 3B and 3C) , suggesting that OspA might not be attached to the membrane. In contrast, unlipidated OspA D17 (LpA D17 ) is trapped within the peptidoglycan layer of the cell wall, maybe attached to the membrane. That is, a 5 min digestion with Lyz exposes a low amount of OspA that is associated with the peptidoglycan layer (Fig. 3B ) and a 45 min or 30 min digestion exposes a high amount of OspA that is associated with the peptidoglycan layer and the membrane (Fig. 3B and 3C) . In order to analyze the potential inflammatory response to the oral administration of L. plantarum expressing OspA lipoprotein, we performed an assay using monolayer cultures of intestinal epithelial cells (T84), a human colon carcinoma cell line, stimulated with UV-killed LpA and LpA D17 and determined the production of IL-8 (Fig. 4) . The co-culture of T84 cells with UVkilled LpA or LpA D17 did not induce significant production of the pro-inflammatory chemokine IL-8 in comparison to the positive control (TNFa).