InlA promotes dissemination of Listeria monocytogenes to the mesenteric lymph nodes during food borne infection of mice

Abstract

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA(m)) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA(m)), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA(m) significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.

Figure 1. Low dose intragastric infection with mouse-adapted L. monocytogenes InlA^m.

Female mice were infected with the indicated inocula by oral gavage and the total number of L. monocytogenes CFU (luminal plus cell-associated) in (A) the small intestine, (B) mesenteric lymph nodes (LN), (D) liver, and (E) spleen was determined 24 hpi. Symbols represent values for individual mice; horizontal lines indicate the mean for each group. Pooled data from 2–3 separate experiments are shown. (C) Mean values +/− SE for L. monocytogenes found in either the luminal contents or cell-associated (adherent plus intracellular) in tissue homogenates 24 h after infection of B6 mice (n = 4) are shown. Dashed horizontal lines indicate the limit of detection for each tissue.

Figure 2. BALB mice are more susceptible than B6 mice to L. monocytogenes infection acquired by ingestion of contaminated food.

Female BALB (white squares) or B6 (black squares) mice were fed 3–5×10⁸ CFU of Lm InlA^m either at noon or 9 PM (night) and the number of L. monocytogenes present in each tissue was determined over time. In panels A, B, E, F, and G, groups of mice were sacrificed 1, 3, 5, and 7 dpi. In panels D, H, and I the indicated organs were harvested 5 dpi. For spleen, liver, small intestines, colon, gall bladder and brain, mean values +/− SD for data pooled from 2 separate experiments (n = 8 mice per group) are shown. Sample groups for fecal analysis in panels (C) and (G) included 8 mice at all time points for night infection and 22 mice (1 dpi), 15 mice (2 dpi), 14 mice (3 dpi), 12 mice (4 dpi), 9 mice (5 dpi), or 4 mice (7 & 8 dpi) for groups infected at noon. Values for BALB mice that were significantly different from the corresponding B6 group by Mann-Whitney analysis are marked with asterisks. Dashed lines indicate the limit of detection.

Figure 3. Female BALB, but not B6 mice, are more susceptible than males to food borne listeriosis.

Groups of mice (n = 4) were fed 5×10⁹ Lm InlA^m at night and bacterial loads were determined 4.5 dpi. Mean values +/− SD from one of two separate experiments are shown. Dashed lines indicate limits of detection.

Figure 4. InlA enhances, but is not required for colonization of the murine intestines.

Female BALB mice were co-infected at night with a 1∶1 mixture of Lm InlA^m and either wild type (wt) Lm EGDe or inlA deletion mutant (ΔinlA) for a total inoculum of 7–9×10⁸ CFU. The total number of each Listeria strain found in either the ileum or colon was determined at both 16 and 60 hpi. Only the terminal third of the small intestine (approximating the ileum) was harvested because a pilot study showed that the majority of L. monocytogenes colonization occurred in the distal portion of the small intestine (Fig. S3). Pooled data from three separate experiments are plotted as competitive indices (CI) to show the ratio of either wt/InlA^m or ΔinlA/InlA^m recovered from each individual mouse. The geometric mean for each group was compared to the theoretical value of 1.0 and the fold difference is shown in parentheses above.

Figure 5. InlA^m enhances spread to the mesenteric lymph nodes and spleen.

Female BALB mice were co-infected at night with a 1∶1 mixture of Lm InlA^m and either wild type (wt) Lm EGDe or an inlA deletion mutant (ΔinlA) for a total inoculum of 5–7×10⁸ CFU and the total number of each Listeria strain found in the mesenteric lymph nodes (MLN), spleen, liver, and gall bladder was determined. Pooled data from at least two separate experiments are shown. In panel A, the data are plotted as competitive indices (CI) to show the ratio of either wt/InlA^m or ΔinlA/InlA^m recovered from each individual mouse at 60 hpi. The geometric mean for each group was compared to the theoretical value of 1.0 and the fold difference is shown in parentheses above. The actual number of CFU recovered in each mouse after wt (triangles) plus InlA^m (squares) co-infection or ΔinlA (circles) plus InlA^m are shown in panels B & C, respectively. Horizontal bars indicate mean values for each group; statistical significance was assessed by student's t test. The limit of detection in each tissue is marked by a dashed line.

Figure 6. L. monocytogenes reside in both extracellular and intracellular compartments in the gut mucosa.

(A) Intestinal tissues were separated into three fractions: the mucus layer (muc), epithelial cells (EC) and the underlying lamina propria (LP) cells. BALB mice (n = 4 per time point) were fed 2×10⁹ Lm InlA^m at night. (B) Mean values +/− SE for the total number of Lm InlA^m in the mucus layer and (C) the number of intracellular (gentamicin resistant) and extracellular (supernatant fraction) Lm InlA^m in the LP and EC layers of the ileum and colon are shown. Data from one of two separate experiments are shown.

Figure 7. InlA^m promotes persistence of L. monocytogenes in the lamina propria of the colon.

Female BALB mice were co-infected at night with a 1∶1 ratio of InlA^m and wild type (wt) Lm EGDe for a total inoculum of 7–9×10⁸ CFU. At 36 and 60 hpi, the ileum and colon from each mouse was fractionated, and the total number of each strain found in the mucus (A, B) and the number of intracellular (Gent^R) Listeria in either epithelial cells (A, C) or lamina propria cells (A, D) was determined. Pooled data (n = 8) from two separate experiments are shown. In panel A, the data are plotted as competitive indices (CI) to show the ratio of either wt/InlA^m recovered from each mouse 60 hpi. The geometric mean for each group was compared to the theoretical value of 1.0 and the fold difference is shown in parentheses above. The actual number of InlA^m CFU (squares) or wt CFU (triangles) recovered from each fraction are shown in panels B, C, and D. Horizontal bars indicate mean values for each group; statistical significance was assessed by student's t test. The limit of detection in each tissue is marked by a dashed line.