Contact-dependent virulence of P. aeruginosa is required for elevating paracellular conductance of cultured pig tracheal epithelia

Poster Session: no. 125This journal suppl. entitled: Special Issue: The 29 Annual North American Cystic Fibrosis Conference October 8-10, 2015Pseudomonas aeruginosa is a common opportunistic pathogen causing severe lung diseases in patients with cystic fibrosis or other airway disorders. In airway, epithelial tight junctions and ion transport are important for defending bacterial infection, whereas it is unclear whether P. aeruginosa modulates these epithelial properties for facilitating its invasion. To address this question, we first characterized our P. aeruginosa ATCC 28573 by a linear relationship between the number of bacteria and OD600 absorbance, doubling time of about 58 minutes for bacterial growth and sequencing specific genes including oprL, ExoA and 16S rDNA. Next, P. aeruginosa in Krebs solution was inoculated to primary culture of pig tracheal epithelia at the multiplicity of infection (MOI) of 0.1, 10 or 200 for 1, 4, 7 and 10 hours. Our Ussing chamber study demonstrates that the basal short-circuit currents (Isc) and Isc changes by different stimulators and inhibitors of ion transporters were not apparently altered by P. aeruginosa inoculation in all MOI groups from 1 to 10 hours, compared to that of the control Krebs solution. However, in all MOI groups tested, the transepithelial conductance (Gt) at basal (Gt-basal) and at the end of experiment (Gt-end) of cultured epithelia were not altered at 1 hour of bacterial inoculation, but slightly increased at 4 hours and greatly elevated at both 7 and 10 hours, compared to that of the control. These data suggest that in cultured epithelia, the tight junctions were loosened by apical P. aeruginosa inoculation in a time-dependent manner. To explore the origin of bacterial toxins that increased Gt, cultured epithelia were inoculated with UV-inactivated P. aeruginosa at MOI = 200 or with the supernatant solution collected from the bacterial solution after 7 hours incubation of P. aeruginosa at MOI = 1000. The data demonstrate that Gt-basal and Gt-end of cultured pig tracheal epithelia were greatly increased by UV-inactivated bacteria but not altered by the supernatant solution. These data suggest that the interactions of bacteria with the epithelial cells may be required for initiating the Gt increase of cultured pig tracheal epithelia. From these results taken together, we speculate that by attaching to the epithelial cells, P. aeruginosa disrupts the assembly of tight junctions at an early stage of infection for the purpose of grabbing nutrients from the mucosal layer or traversing through the epithelial barrier