Expulsion of E. coli from infected BECs is cAMP dependent. (A) Intracellular cAMP levels were determined by a cAMP enzyme immunoassay either before or 1 or 4 h after E. coli ORN103(pSH2) infection of 5637 BECs. At the same time, the number of exocytosed E. coli ORN103(pSH2) was determined by sampling the extracellular media 1 and 4 h after gentamicin treatment. The increase in extracellular E. coli correlated to the increase in intracellular cAMP. (B) Control BECs and AC3 kd BECs were infected for 1h with E. coli ORN103(pSH2). After a 30-min gentamicin treatment, the E. coli ORN103(pSH2) infected BECs were incubated with a fresh medium containing bacteriostatic agents and D-mannose to prevent bacterial growth and reentry of bacteria, respectively. After 4 h, the medium was cultured for extracellular E. coli ORN103(pSH2), and revealed that AC3 kd BECs were significantly less effective in expelling intracellular E. coli ORN103(pSH2) than the control BECs. *, P < 0.05 by unpaired t test when compared with uninfected controls. Error bars represent SD (A) and SEM (B).

E. coli exocytosis by BECs depends on Rab27b and caveolin-1. (A) Cell extracts from uninfected (Uninfected) and E. coli ORN103(pSH2)-infected (E. coli) BECs were fractionated on a sucrose gradient. Rab27b and caveolin-1 were distributed in fractions 5 to 8 in uninfected BECs, and after infection, the majority of these proteins had shifted into fraction 5. (B) A pull-down assay showing specific coassociation between Rab27b and caveolin-1. The assays using GFP-Rab27b expressing BECs (GFP-Rab27b), but not control cells expressing only GFP (GFP Ctrl) detected caveolin-1 as a binding partner. UI, uninfected; EC, E. coli infected. (C) Indirect immunofluorescence showed colocalization between caveolin-1 (red) and Rab27b (green). (D) Rab27b and/or caveolin-1 expression levels in regular BECs were reduced by using gene specific siRNAs. Compared with levels in sham siRNA transfected cells (Ctrl), protein expression levels of Rab27b and caveolin-1 were reduced by ≈57 and ≈80% in kd cells (siRNA), respectively (Inset). A significant decrease in bacterial exocytosis in caveolin-1 kd BECs (Cav1 siRNA), in Rab27b kd BECs (Rab27b siRNA), and in double kd BECs (Cav1/Rab27b siRNA) was observed compared with controls. *, P < 0.03 by unpaired t test when compared with negative control siRNA transfected BECs. Error bars represent SEM.

E. coli exocytosis from infected BECs. (A) The 5637 BECs were infected with various bacteria for 1 h, after which a standard gentamicin protection assay was performed to quantify the number of intracellular bacteria (0 h). A significant decrease in intracellular E. coli ORN103(pSH2) and E. coli CI5 was seen at 24 h after the addition of gentamicin (24 h). Unlike E. coli ORN103(pSH2) and E. coli CI5, the numbers of intracellular S. typhimurium SL1344 and S. aureus 54 were significantly increased at 24 h after initial infection and after gentamicin treatment. (B) Intracellular and extracellular E. coli ORN103(pSH2) numbers in BECs at 0 and 4 h after gentamicin treatment; 5637 BECs were infected with 1000 MOI E. coli ORN103(pSH2). The sum of numbers of intracellular and extracellular E. coli at 4 h after gentamicin treatment was similar to intracellular bacteria numbers at 0 h, suggesting bacterial exocytosis from infected BECs. (C) Treatment of infected BECs with NH₄Cl and Bafilomycin, which neutralize bactericidal activity within lysosomes, caused no change of numbers of intracellular bacteria, indicating the compartment harboring E. coli did not possess bactericidal activity. (D) Quantitative E. coli exocytosis assays were performed by using human primary BECs and a clinical isolate of UPEC strain E. coli CI5. Significant numbers of intracellular E. coli CI5 were expelled from infected primary BECs at 4 h after the gentamicin treatment. Bars represent the mean + SEM. *, P < 0.05; **, P < 0.03; ***, P < 0.001, relative to Intracellular E. coli titers at 0 h in A, B, and D.

E. coli expulsion from infected BECs is initiated when TLR4 interacts with bacterial LPS. (A) WT BECs were infected for 1h with E. coli ORN103(pSH2). When cells were treated with gentamicin, 100 μg/mL LPS was added to the culture medium to examine the effect on bacterial exocytosis, when indicated. After a 30-min gentamicin and LPS treatment, infected BECs were incubated with fresh media containing bacteriostatic agents and D-mannose. After 4 h, the medium was cultured for extracellular E. coli. Treatment of BECs with LPS resulted in a significant increase in E. coli ORN103(pSH2) exocytosis compared with untreated BECs (Untreated). (B) Exocytosis assays were performed using control and TLR4 kd BECs as described above except LPS treatment. TLR4 kd BECs were significantly less effective in expelling intracellular E. coli than the control BECs. *, P < 0.05 by unpaired t test when compared with controls. Error bars represent SEM. (C) Human primary BECs were used for exocytosis assays. Primary BECs were infected with either WT E. coli (E. coli W3110) or LPS mutant E. coli (a msbB mutant E. coli MLK1067). When indicated, LPS or MPL were added to the media as described before. **, P < 0.0005 by unpaired t test when compared with WT E. coli infected BECs. Error bars represent SD.

E. coli expulsion by BECs requires MyRIP activity. (A) A pull-down assay was performed using Rab27-, MyRIP-, and PKA RII-specific antibodies. A control immunoblot revealed that equal levels of caveolin-1 protein were expressed in the cell extracts before and after infection (Inset). All these signaling molecules were coassociated with caveolin-1 in BECs even before infection (UI) and after infection (EC), the coassociation became markedly more increased. (B) An E. coli exocytosis assay was performed to examine effects of MyRIP on bacteria expulsion. MyRIP mRNA levels were reduced by ≈40% in kd BECs (Inset), and levels of E. coli exocytosis were significantly dropped by 40%. *, P < 0.002 by unpaired t test when compared with control siRNA transfected BECs. Error bars represent SEM.