Fig. 2

Cell surface expression and secretion of Hcp protein by rL. lactis. A Representative immunofluorescence images of nisin-induced rL. lactis cells surface expressing Hcp probed with rabbit polyclonal anti-Hcp antibody (1:50 dilution), and FITC conjugated goat anti-rabbit IgG secondary antibody (1:500 dilution). Un-induced rL. lactis and empty L. lactis (NZ000) cells served as control, showing no fluorescence signal. Images were acquired by an inverted fluorescence microscope at 60X magnification. All the images were analyzed using ImageJ software and shown at a scale bar of 10 µm (a–c). Data represent mean fluorescence intensity (MFI) of L. lactis cells ± SE of three independent experiments. Asterisks indicate a statistically significant increase in the MFI of nisin-induced rL. lactis cells (**P ≤ 0.01) with respect to the empty NZ9000 cells (d). Flow cytometric analysis of nisin induced L. lactis cells showing a significant shift of stained rL. lactis population (blue line) as compared to the controls (e). Data represent MFI of L. lactis cells ± SE of three independent experiments. Asterisks indicate a statistically significant difference (*P ≤ 0.05) compared to the control (f). B Representative well images indicates specific detection of secreted Hcp in the culture supernatant of induced rL. lactis cells harbouring pSEC-spUSP45-hcp plasmid by rabbit polyclonal anti-Hcp antibody using indirect ELISA. Recombinant Hcp expressed in E. coli, and native Hcp secreted by laboratory isolate of C. jejuni (18aM) served as a positive control, while empty L. lactis cells (NZ9000) were kept as a negative control (a). Data represent the mean absorbance (A450) ± SE of quadruplicates samples. Asterisks indicate a statistically significant difference (**P ≤ 0.01) with respect to the control (empty NZ9000) (b). Dot blot images showing specific detection of secreted Hcp in the culture supernatant of induced rL. lactis cells by rabbit polyclonal anti-Hcp antibody (c)