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.

Role of p38 MAP Kinase and Transforming Growth Factor-β Signaling in Transepithelial Migration of Invasive Bacterial Pathogens

“…It was also suggested that the ability of H. influenzae to penetrate the epithelial layer protects them from antibiotics and antibody-mediated bactericidal activity (25). Burkholderia cenocepacia also disrupts a differentiated epithelial barrier, an event associated with loss of occludin from tight junctions (26).…”

Rhinovirus-Induced Barrier Dysfunction in Polarized Airway Epithelial Cells Is Mediated by NADPH Oxidase 1

“…After 90 min of incubation, infection medium from the apical surface was replaced with fresh growth medium, and the cells were further incubated for 4 to 24 h at 33°C. In inhibition studies, N-propyl gallate, diphenylene iodonium (DPI), oxypurinol, quercetin (all from SigmaAldrich, St. Louis, MO), or the Rac1 inhibitor NSC23766 (EMD Chemicals, Gibbstown, NJ) was added to both the apical and basolateral chambers 90 min after RV infection and incubated for 24 h, and R T was measured with an Evom voltmeter equipped with an EndOhm 6 tissue resistance measurement chamber (World Precision Instruments, Sarasota, FL) (25,41).…”

Bacillus cereusInduces Permeability of an In Vitro Blood-Retina Barrier

“…After coating, Transwells and coverslips were washed twice with phosphate-buffered saline (PBS) and RPE cells were seeded at 2 ϫ 10 5 /ml in DMEM/F12 supplemented with 5% FBS and 1% GLN. Medium was removed from the apical chamber at 48 to 72 h postseeding to create an air-liquid interface and to stimulate polarization (day 0) (33). Polarized monolayers were incubated for 15 days before infection.…”