Translocation of Yersinia enterocolitica across reconstituted intestinal epithelial monolayers is triggered by Yersinia invasin binding to beta1 integrins apically expressed on M-like cells

Abstract

SummaryYersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed b1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-b1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of b1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria.

Characterization of antigen and bacterial transport in the follicle-associated epithelium of human ileum

“…Mechanisms of uptake and transport were studied by microscopy and by pharmacological inhibition studies in human FAE tissue, as well as in the in vitro lymphocyte-epithelial cell coculture model of human FAE initially described by Kerneís et al, 11 and subsequently used by several groups to study FAE and M-cell function. [12][13][14][15][16][17][18] Our findings show that human ileal FAE is not only structurally, but also functionally distinct from regular VE, with enhanced transport of antigens and bacteria into the underlying lymphoid tissue.…”

“…In a paper by Kerneís et al 11 it was shown that coculture of Peyer's patch lymphocytes and intestinal epithelial cells may trigger conversion of the epithelial cells to M cells. This model and variants of it have been used to study M-cell function, [12][13][14][15][16][17] and by using our previously established modification 18 of the original model, mechanisms of antigen and bacterial uptake could be studied. Briefly, intestinal epithelial Caco-2 cells were grown on Matrigelt (Becton Dickinson, USA) coated polycarbonate filters (Costar, Baedvenhorp, NL, USA) with a mean pore size of 3.0 mm.…”

“…Before experiments, the cell monolayers were incubated with either the amiloride analog 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), (100 mM), an inhibitor of the apical membrane Na þ /H þ exchanger, which has been used as an inhibitor of macropinocytosis, 23,24 12,25 Transepithelial resistance was measured before and after incubation. For HRP studies, 10 À5 M HRP, with or without EIPA, was added to the apical side of the cells, samples were withdrawn at regular time intervals, stored at À201C and analyzed as described for human tissue.…”

Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis

“…To determine the number of intracellular bacteria, a gentamicin protection assays was performed as described earlier. 28 Briefly, cultures were washed with PBS and incubated for 1 h in the presence of gentamicin (100 mg/ml) to kill remaining extracellular bacteria. Cells were lysed with 1% Triton X-100 in PBS, and intracellular viable bacteria assessed by plating serial 10-fold dilutions on plates with kanamycin (50 mg/ml) and ampicillin (100 mg/ml).…”

“…Together, these two proteins mediate initial adhesion, uptake and translocation of Yersinia across the intestinal epithelium. 26,27 Invasin is known to exert its effect via a high-affinity interaction with b1-integrin, which is expressed on the luminal surface of M-cells (epithelial cell type specialized in antigen and bacterial uptake), 28,29 but only on the basolateral aspect of villus epithelial cells.…”