Aeromonas sobria serine protease decreases epithelial barrier function in T84 cells and accelerates bacterial translocation across the T84 monolayer in vitro

Abstract

Aeromonas sobria is a pathogen causing food-borne illness. In immunocompromised patients and the elderly, A. sobria can leave the intestinal tract, and this opportunistically leads to severe extraintestinal diseases including sepsis, peritonitis, and meningitis. To cause such extraintestinal diseases, A. sobria must pass through the intestinal epithelial barrier. The mechanism of such bacterial translocation has not been established. Herein we used intestinal (T84) cultured cells to investigate the effect of A. sobria serine protease (ASP) on junctional complexes that maintain the intercellular adhesion of the intestinal epithelium. When several A. sobria strains were inoculated into T84 monolayer grown on Transwell inserts, the strain with higher ASP production largely decreased the value of transepithelial electrical resistance exhibited by the T84 monolayer and markedly caused bacterial translocation from the apical surface into the basolateral side of T84 monolayer. Further experiments revealed that ASP acts on adherens junctions (AJs) and causes the destruction of both nectin-2 and afadin, which are protein components constituting AJs. Other studies have not revealed the bacterial pathogenic factors that cause the destruction of both nectin-2 and afadin, and our present results thus provide the first report that the bacterial extracellular protease ASP affects these molecules. We speculate that the destruction of nectin-2 and afadin by the action of ASP increases the ability of A. sobria to pass through intestinal epithelial tissue and contributes to the severity of pathological conditions.

Fig 1. The ASP activity is closely related to destruction of intestinal epithelial barrier function.

A: T84 cells were cultured in a Transwell system and then infected with several types of A. sobria strains. After 3 hr of infection (MOI = 5), the reduction in TER was measured. The TER value at 0 hr of infection was taken as 100%. NT: The TER value was measured without bacterial infection. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01. B: The ability of the A. sobria strains to translocate across the intestinal epithelial cells (T84 cells) at 6 hr after infection (MOI = 5) was assessed using quantitative cultures of medium obtained from the lower chambers. NT: The experiment was done without bacterial infection. ND: The bacterial translocation could not be detected in this experimental condition. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: The proteolytic activity in the culture supernatant of each A. sobria strain was measured as described in the text. The experiments were performed in triplicate. The data are mean ± SD (error bars). D: The presence of ASP in the culture supernatant of each strain was immunologically detected by a western blotting analysis as described in the text.

Fig 2. The involvement of ASP in the translocation of A. sobria across intestinal epithelial cell monolayer.

A: The asp gene of A. sobria 288 strain was knocked out. The immunological analysis using western blotting revealed that the asp-knocked-out strain (#288 ΔASP) did not produce ASP and the complemented strain (#288 ΔASP::ASP) produced ASP again. B: The proteolytic activity in the culture supernatant of each strain. The experiments were performed in triplicate. The data are mean ± SD (error bars). C: T84 cells were cultured in a Transwell system and then infected with the wild-type A. sobria strain (#288), the asp-knocked-out strain (#288 ΔASP), or the complemented strain (#288 ΔASP::ASP). After 6 hr of infection (MOI = 5), the ability of these A. sobria strains to translocate across the T84 cell monolayer was assessed in the same way as that described in the Fig 1 legend. The experiments were performed in triplicate. The data are mean ± SD (error bars). *p<0.01, **p<0.05.

Fig 3. ASP caused disruption of the intestinal epithelial barrier.

A: T84 cells were cultured in a Transwell system, and the TER value was measured in the presence of various concentrations (nM) of ASP or absence (NT) of ASP. We also examined the effect of the serine protease inhibitor PMSF on the action of ASP. B: The passive diffusion of FITC-labeled dextran molecules across the T84 monolayer (from the apical side to the basolateral side) treated with ASP was measured. All experiments were performed in triplicate. The data are mean ± SD (error bars).

Fig 4. Target proteins on which ASP acts.

T84 cells were treated with (nM) or without (NT) various concentrations of ASP before the extraction. After the extraction, we detected the proteins constituting the junctional complexes by using a specific antibody against each protein shown in the figure. The results of quantitative analysis of the amount of blotted protein are also shown below the image of western blotting. These experiments were performed in triplicate. The data are mean ± SD (error bars).

Fig 5. Immunohistological analysis of the effects of ASP.

T84 cells were treated with or without (NT) 500 nM ASP for 6 hr. The cells were reacted with a specific antibody against nectin-1, nectin-2, and E-cadherin and visualized using the secondary antibody conjugated with a fluorescent substance, Cy5 or FITC. Nuclei were stained with PI. The merged images are shown in each panel. Merge 1: nectin-2 and E-cadherin, Merge 2: afadin and E-cadherin, and Merge 3: nectin-1 and E-cadherin. Z: Z-stack showing entire sample volume image was also shown.

Fig 6. The effects of ASP on recombinant nectin-2.

A: Schematic representation of nectin-2. B: Schematic representation of the recombinant nectin-2 (rNectin-2) used in this study. C: The rNectin-2 preparation was treated with various concentrations of ASP (nM). The reaction mixture was then subjected to SDS-PAGE. Several protein fragments thought to be caused by the action of ASP were analyzed. D: Cleavage of fluorogenic peptide substrates by ASP. K_cat/K_m values were calculated from the reaction of ASP with the substrates. The experiments were performed in triplicate. The data are mean ± SD (error bars).

Fig 7. The translocation of A. sobria strains across intestinal epithelial cells.

A: T84 cells were infected (MOI = 1) with or without (NT) A. sobria strain 120, 123 or 288. Bacterial internalization was confirmed using the Aeromonas-specific probe FITC-AER66 as described in Materials and Methods (fluorescence image). The figure shown as ‘Merge’ is a superposition of the fluorescence image (left side) and the blight image (center). B: T84 cells were infected (MOI = 1) with A. sobria strain 120, 123 or 288. The number of bacteria survived from gentamicin protection assay was determined and indicated as colony forming unit (CFU). The experiments were performed in triplicate and the data are means ± SD (error bars).