Fig. 9. OmpA⁺ E. coli interaction with HBMEC induces NO mediated cGMP production, thereby enhancing the expression of Ec-gp96 for increased invasion.

(A) Confluent monolayers of HBMEC were infected with OmpA⁺ or OmpA⁻ E. coli for varying periods, total cell lysates were prepared and the levels of cGMP were determined as described in the methods section. In some experiments, the monolayers were pretreated with AG, ODQ or 8-Br-cGMP prior to infection with the bacteria. (B) HBMEC were seeded onto Transwell filters and treated as described in A and the TEER was measured. (C) HBMEC were either untreated or infected with OmpA⁺ or OmpA⁻ E. coli or treated with 8-Br-cGMP or ODQ for 1 h. The cells were then subjected to flow cytometry analysis after staining with anti-Ec-gp96 antibodies. (D) HBMEC monolayers pretreated with ODQ or 8-Br-cGMP were subjected to binding and invasion assays using OmpA⁺ E. coli. The results are expressed as percent binding or invasion considering the binding/invasion of control HBMEC as 100%. All these experiments were performed at least three times in triplicates and presented as mean ± SD. The increase or decrease in the expression of Ec-gp96, binding to or invasion was significant compared to control HBMEC (*P<0.001 by Student’s t test).