Salmonella Effector SpvB Disrupts Intestinal Epithelial Barrier Integrity for Bacterial Translocation

Abstract

Salmonella are common enteric bacterial pathogens that infect both humans and animals. Intestinal epithelial barrier, formed by a single layer of epithelial cells and apical junctional complex (AJC), plays a crucial role in host defense against enteric pathogens to prevent bacterial translocation. However, the underlying mechanisms of intestinal epithelial barrier dysfunction caused by Salmonella are poorly understood. It is found that a locus termed Salmonella plasmid virulence (spv) gene exists extensively in clinically important Salmonella serovars. SpvB is a key effector encoded within this locus, and closely related to Salmonella pathogenicity such as interfering with autophagy and iron homeostasis. To investigate the interaction between SpvB and intestinal epithelial barrier and elucidate the underlying molecular mechanism, we used the typical foodborne disease agent Salmonella enterica serovar Typhimurium (Salmonella typhimurium) carrying spvB or not to construct infection models in vivo and in vitro. C57BL/6 mice were orally challenged with S. typhimurium wild-type strain SL1344 or spvB-deficient mutant strain SL1344-ΔspvB. Caco-2 cell monolayer model, as a widely used model to mimic the human intestinal epithelium in vitro, was infected with SL1344, SL1344-ΔspvB, or spvB complementary strain SL1344-c-ΔspvB, respectively. The results showed that SpvB enhanced bacterial pathogenicity during S. typhimurium infection in vivo, and contributed to intestinal epithelial barrier dysfunction in both infection systems. This SpvB-mediated barrier dysfunction was attributed to the cellular redistribution of Claudin-1, Occludin, and E-cadherin junctional proteins. Moreover, by using pharmacological inhibitors, we found that F-actin rearrangement and suppression of protein kinase C (PKC) signaling pathway were involved in SpvB-mediated barrier dysfunction. In conclusion, the study reveals the contribution of Salmonella effector SpvB to the dysfunction of intestinal epithelial barrier integrity, which facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. Our findings broaden the understanding of host-pathogen interactions in salmonellosis, and provide new strategies for the therapy in limiting bacterial dissemination during infection.

Keywords: F-actin; Salmonella; SpvB; apical junctional complex; intestinal epithelial barrier; protein kinase C.

Figure 1

SpvB enhances bacterial pathogenicity in S. typhimurium infection. Streptomycin-pretreated mice were orally infected with the indicated S. typhimurium strains (1 × 10⁸ CFUs) for 48 h. (A–D) Salmonella counts (per gram of the sample) in liver (A), spleen (B), MLN (C), and colon (D) of infected mice (n = 5). (E) Representative H&E-stained images (scale bar, 50 µm) of liver, spleen and colon tissues from uninfected (control) mice or Salmonella-infected mice. Data are presented as the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

Figure 2

SpvB contributes to intestinal barrier dysfunction and bacterial dissemination in vivo. Streptomycin-pretreated mice were orally infected with the indicated S. typhimurium strains (1 × 10⁸ CFUs) for 48 h. (A) 4 kDa FITC-dextran permeability through the intestinal epithelium of uninfected (control) or Salmonella-infected mice in serum. (B–E) Salmonella counts (per gram of colon) in mucus layer (B), epithelial cells (C), LP cells (D) and extracellular space (supernatant fraction, E) of the epithelial cell and LP layers from the colon tissue of infected mice (n = 5). Data are presented as the mean ± SEM. **P < 0.01; *P < 0.05; ns, not significant.

Figure 3

SpvB contributes to intestinal barrier dysfunction in vitro. (A-C) Caco-2 cells were cultured on Transwell inserts to form cell monolayers, and treated with the indicated S. typhimurium strains at an MOI of ~100. TEER (A) was measured at the indicated times. Significant difference vs. SL1344 (**P < 0.01; *P < 0.05). Significant difference vs. SL1344-c-ΔspvB (^##P < 0.01; ^#P < 0.05). 4 kDa FITC-dextran flux (B) and bacterial translocation (C) across Caco-2 monolayers were measured 3 h.p.i. (D, E) Caco-2 cells were infected with the indicated S. typhimurium strains at an MOI of ~100. Total number of associated Salmonella (D, Salmonella adhesion) and the number of internalized Salmonella (F, Salmonella invasion) were determined. Data are presented as the mean ± SEM. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

Figure 4

SpvB induces junctional protein dysregulation in Salmonella infection. (A–D) Streptomycin-pretreated mice were orally infected with the indicated S. typhimurium strains (1 × 10⁸ CFUs) for 48 h. (A) Western blot analysis and densitometry plots of AJC proteins of colonic IECs from infected mice. (B–D) Images of the mouse colon tissue sections showing localization of Claudin-1 (B, red), Occludin (C, red) and E-cadherin (D, red). Nuclei, blue. Scale bar, 10 µm. (E–G) Caco-2 cells were treated with the indicated S. typhimurium strains at an MOI of ~100. Western blot analysis and densitometry plots of AJC proteins from the detergent-insoluble fraction (E, membrane) and detergent-soluble fraction (F, cytosolic) of Caco-2 cells 1 h and 3 h.p.i. (G) Images of Caco-2 cells showing membrane localization of Claudin-1 (red), Occludin (red) and E-cadherin (red) 3 h.p.i. Scale bar, 50 µm. Data are presented as the mean ± SEM. *P < 0.05.

Figure 5

F-actin rearrangement is responsible for SpvB-mediated barrier dysfunction. Caco-2 cells were pre-treated with the inhibitor of actin polymerization, Cytochalasin D (2 µM), 1 h prior to infection with the indicated S. typhimurium strains. 4 kDa FITC-dextran flux (A) and bacterial translocation (B) across Caco-2 monolayers were measured 3 h.p.i. (C, D) Western blot analysis and densitometry plots of AJC proteins from the detergent-insoluble fraction (C, membrane) and detergent-soluble fraction (D, cytosolic) of Caco-2 cells 3 h.p.i. Data are presented as the mean ± SEM. ns, not significant.

Figure 6

Down-regulated PKC activity is involved in SpvB-mediated barrier dysfunction. (A) Caco-2 cells were treated with the indicated S. typhimurium strains at an MOI of ~100. Western blot analysis and densitometry plots showing the activation of PKC. (B) 293T cells were transfected with pEGFP-N1-SpvB or pEGFP-N1 for 24 h. Western blot analysis showing the expression of HA-SpvB and the activation of PKC. (C–F) Caco-2 cells were pre-treated with the inhibitor of PKC activation, Bisindolylmaleimide I (1 µM), 1 h prior to infection with the indicated S. typhimurium strains. 4 kDa FITC-dextran flux (C) and bacterial translocation (D) across Caco-2 monolayers were measured 3 h.p.i. (E, F) Western blot analysis and densitometry plots of AJC proteins from the detergent-insoluble fraction (E, membrane) and detergent-soluble fraction (F, cytosolic) of Caco-2 cells 3 h.p.i. Data are presented as the mean ± SEM. *P < 0.05; ns, not significant.

Figure 7

General scheme of SpvB-mediated bacterial translocation during Salmonella infection. Salmonella effector SpvB released into the intestinal epithelial cell could lead to F-actin rearrangement and suppression of PKC signaling pathway, which contributes to the cellular redistribution of Claudin-1, Occludin and E-cadherin junctional proteins. This SpvB-mediated dysfunction of intestinal epithelial barrier integrity thereby facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. AJ, adherens junction; AJC, apical junctional complex; IEC, intestinal epithelial cells; PKC, protein kinase C; SCV, Salmonella-containing vacuole; TJ, tight junction.