Disruption of tight junctions during traversal of the respiratory epithelium by Burkholderia cenocepacia

Abstract

Burkholderia cenocepacia is an opportunistic bacterial species capable of causing life-threatening respiratory tract infection in persons with cystic fibrosis (CF). Unlike most other pathogens in CF, which typically remain confined to the endobronchial spaces, B. cenocepacia can traverse airway epithelium to cause bacteremia and sepsis. The mechanisms by which this occurs, however, are unknown. We examined the transmigration of B. cenocepacia through polarized respiratory epithelium. Representatives of three "epidemic" lineages common among CF patients in North America were able to traverse polarized 16HBE14o- cells in vitro. Transmigration of bacteria was associated with significant perturbations in epithelial permeability, as measured by a loss of transepithelial electrical resistance and increased flux of bovine serum albumin across the cell layer. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and trypan blue exclusion assays, as well as lactate dehydrogenase levels, did not indicate excessive cytotoxicity or cell death in infected cell layers. Rather, confocal fluorescence microscopy demonstrated the loss of occludin from tight junctions. In contrast, zonula occludens 1 was well preserved along intercellular borders. Western blot analysis showed a shift in the major occludin isoforms from high- to low-phosphorylation states during infection. These observations suggest that B. cenocepacia traverses polarized respiratory epithelium by the dephosphorylation and dissociation of occludin from the tight-junction complex.

FIG. 1.

Translocation of bacteria across polarized 16HBE cell layers. Cell layers were uninfected (○) or infected with E. coli JM109 (•), E. coli E2348/69 (⋄), B. cenocepacia PC8 (⧫), B. cenocepacia PC184 (□), or B. cenocepacia AU0355 (▴). The data points represent the mean (± standard error of the mean) concentrations of bacteria in basolateral media of four replicate experiments at each time point. The data were transformed to log₁₀ and analyzed by one-way ANOVA with Tukey-Kramer posttest analysis. At the 8-h time point, concentrations of AU0355 and PC184 were significantly greater than that of PC8 (P < 0.05).

FIG. 2.

Integrity of infected and uninfected polarized 16HBE cell layers. (a) TER across cell layers. Cell layers were uninfected (○) or infected with E. coli JM109 (•), B. cenocepacia PC8 (⧫), B. cenocepacia PC184 (□), or B. cenocepacia AU0355 (▴). Uninfected and JM109-infected cell layers show significantly less reduction in TER than B. cenocepacia-infected cell layers (P < 0.001). The data are presented as percent change (± standard error of the mean) from the initial TER at each time point indicated. (b) Permeability of cell layers. P_app were calculated by measuring the flux of FITC-BSA from apical to basolateral media. Statistically significant differences (determined by using one-way ANOVA with Tukey-Kramer posttest analysis) between uninfected (control) cell layers or cell layers infected with E. coli JM109 and B. cenocepacia-infected cell layers are indicated by asterisks. *, P < 0.05; ***, P < 0.001.

FIG. 3.

Confocal immunofluorescence microscopy of infected polarized 16HBE cell layers. (a) Uninfected cell layer immunostained with antibody to ZO-1. (b) Uninfected cell layer immunostained with antibody to occludin. (c) Cell layer infected with AU0355 for 24 h and then immunostained with antibodies to occludin (green) and B. cenocepacia (red); the white arrows indicate occludin dislocated to cytoplasm. (d) Z-series cross-section of entire cell layer depicted in panel c; the black arrows point to the apical surface and indicate areas of colocalization of bacteria and occludin.

FIG. 4.

Western blot analysis of detergent-insoluble protein fractions from 16HBE cells. Equal amounts of protein were loaded in each lane, and the blots were probed with mouse monoclonal antibody to occludin (A), ZO-1 (B), or β-actin (C). Lane 1, molecular weight markers; lane 2, mock-infected control cells; lane 3, cells infected with noninvasive E. coli JM109 for 24 h; lanes 4 and 5, cells infected with B. cenocepacia AU0355 for 8 h and 24 h, respectively; lanes 6, 7, and 8, cells infected with enteropathogenic E. coli S2348/69 for 3, 6, and 12 h, respectively. All infections were done with an MOI of 20:1. The solid arrow indicates the position of hyperphosphorylated occludin; the open arrow indicates the position of relatively dephosphorylated occludin.