Wine Pomace Product Inhibit Listeria monocytogenes Invasion of Intestinal Cell Lines Caco-2 and SW-480

Abstract

Red wine pomace products (WPP) have antimicrobial activities against human pathogens, and it was suggested that they have a probable anti-Listeria effect. This manuscript evaluates the intestinal cell monolayer invasive capacity of Listeria monocytogenes strains obtained from human, salmon, cheese, and L. innocua treated with two WPP (WPP-N and WPP-C) of different polyphenol contents using Caco-2 and SW480 cells. The invasion was dependent of the cell line, being higher in the SW480 than in the Caco-2 cell line. Human and salmon L. monocytogenes strains caused cell invasion in both cell lines, while cheese and L. innocua did not cause an invasion. The phenolic contents of WPP-N are characterized by high levels of anthocyanin and stilbenes and WPP-C by a high content of phenolic acids. The inhibitory effect of the WPPs was dependent of the strain and of the degree of differentiation of the intestinal cells line. The inhibition of Listeria invasion by WPPs in the SW480 cell line, especially with WPP-C, were higher than the Caco-2 cell line inhibited mainly by WPP-N. This effect is associated with the WPPs' ability to protect the integrity of the intestinal barrier by modification of the cell-cell junction protein expression. The gene expression of E-cadherin and occludin are involved in the L. monocytogenes invasion of both the Caco-2 and SW480 cell lines, while the gene expression of claudin is only involved in the invasion of SW480. These findings suggest that WPPs have an inhibitory L. monocytogenes invasion effect in gastrointestinal cells lines.

Keywords: Caco-2; E-cadherin; Listeria monocytogenes; SW480; claudin; occludin; polyphenols; red wine pomace; virulence.

Figure 1

Total antioxidant capacity of the studied wine pomace products (WPP) evaluated using the Quencher method (Q-) of the classical Folin–Ciocalteu (FC) (A), Q-ABTS (B), and Q-FRAP (C) assays. GAE: gallic acid equivalents; TE: Trolox equivalents. WPP-N: Wine pomace product of the north winery; WPP-C: Wine pomace product of the center winery. Results are expressed as the mean ± standard deviation (n = 3). * Indicates statistical higher values (p < 0.05).

Figure 2

Caco-2 cell junction protein expression after L. monocytogenes invasion. Values represent the relative gene expression of claudin (A), occludin (B), and E-cadherin (C) of the control cells (cells uninfected with L. monocytogenes) and cells infected with ILSI9, ILSI17, and ILSI18 (clinical L. monocytogenes strains) and S11 and E10.652 (food L. monocytogenes strains) and L. innocua. The results are presented as the means ± SD (n = 4). Different letters on each column indicate significant differences (ANOVA, p < 0.05) among the mean values.

Figure 3

SW480 cell junction protein expression after L. monocytogenes invasion. Values represent the relative gene expression of claudin (A), occludin (B), and E-cadherin (C) of the control cells (cells uninfected with L. monocytogenes) and cells infected with ILSI9, ILSI17, and ILSI18 (clinical L. monocytogenes strains) and S11 and E10.652 (food L. monocytogenes strains) and L. innocua. The results are presented as the means ± SD (n = 4). Different letters on each column indicate significant differences (Student’s t-test, p < 0.05) among the mean values.

Figure 4

Effect of WPP on the virulence of ILSI9, ILSI17 ILSI18, and S11 L. monocytogenes strains on Caco-2 cells. (A) In vitro invasion capacity of the L. monocytogenes strains treated with WPP-N, expressed as % of inhibition with respect to each control (invasion capacity of each Listeria strain grown in the absence of WPP-N). (B–D) Relative values of the gene expression of claudin (B), occludin (C), and E-cadherin (E) with respect to the corresponding control (cells infected with each L. monocytogenes strain grown in the absence of WPP-N). (E) In vitro invasion capacity of the Listeria strains treated with WPP-C, expressed as % of inhibition with respect to each control (invasion capacity of each Listeria strain grown in the absence of WPP-C). (F–H) Relative values of the gene expression of claudin (E), occludin (F), and E-cadherin (G) with respect to the corresponding control (cells infected with each L. monocytogenes strain grown in the absence of WPP-C). Gene expression was assessed by real-time PCR. Results are presented as the means ± SD (n = 4). Columns marked with * indicate gene expression values statically different than the respective control (Student’s t-test, p < 0.05). Letters on each column indicate significant differences among values for each Listeria strain (ANOVA, p < 0.05).

Figure 5

Effect of WPP on the virulence of ILSI9, ILSI17 ILSI18, and S11 L. monocytogenes strains on SW480 cells. (A) In vitro invasion capacity of L. monocytogenes strains treated with WPP-N, expressed as % of inhibition with respect to each control (invasion capacity of each Listeria strain grown in the absence of WPP-N). (B–D) Relative values of the gene expression of claudin (B), occludin (C), and E-cadherin (E) with respect to the corresponding controls (cells infected with each L. monocytogenes strain grown in the absence of WPP-N). (E) In vitro invasion capacity of the Listeria strains treated with WPP-C, expressed as % of inhibition with respect to each control (invasion capacity of each Listeria strain grown in the absence of WPP-C). (F–H) Relative values of the gene expression of claudin (E), occludin (F), and E-cadherin (G) with respect to the corresponding control (cells infected with each L. monocytogenes strain grown in the absence of WPP-C). Gene expression was assessed by real-time PCR. The results are presented as the means ± SD (n = 4). Columns marked with * indicate gene expression values statically different than the respective control (Student’s t-test, p < 0.05). Letters on each column indicate significant differences among the values for each Listeria strain (p < 0.05).