Pseudomonas aeruginosa Homoserine Lactone Activates Store-operated cAMP and Cystic Fibrosis Transmembrane Regulator-dependent Cl− Secretion by Human Airway Epithelia

Abstract

The ubiquitous bacterium Pseudomonas aeruginosa frequently causes hospital-acquired infections. P. aeruginosa also infects the lungs of cystic fibrosis (CF) patients and secretes N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12) to regulate bacterial gene expression critical for P. aeruginosa persistence. In addition to its effects as a quorum-sensing gene regulator in P. aeruginosa, 3O-C12 elicits cross-kingdom effects on host cell signaling leading to both pro-or anti-inflammatory effects. We find that in addition to these slow effects mediated through changes in gene expression, 3O-C12 also rapidly increases Cl ؊ and fluid secretion in the cystic fibrosis trans-

Deleterious impact ofPseudomonas aeruginosaon cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells

“…Furthermore, BUYCK et al [35] have shown that P. aeruginosa LPS-stimulated Cl − currents through CFTR channels in 16HBE14o-cells, probably via a calcium signal. Similarly, N-(3-oxo-dodecanoyl)-S-homoserine lactone (3O-C12), a quorum sensing molecule secreted by P. aeruginosa, has been shown to rapidly enhance Cl − and fluid secretion as well as cytosolic calcium and cAMP levels [36]. It has also been reported that P. aeruginosa flagellin activated CFTR-dependent anion secretion in Calu-3 (through p38 signalling) as well as in primary cultures of human bronchial epithelial cells [37].…”

Thapsigargin blocks Pseudomonas aeruginosa homoserine lactone-induced apoptosis in airway epithelia

“…In contrast, in the presence of Tg ϩ C12, Ca er is low but Ca mito (Fig. 8) and Ca cyto (30) are higher than control, and C12-triggered apoptosis is blocked. Because Ca 2ϩ -free solution and/or Ca 2ϩ -free solution ϩ BAPTA-AM were inhibitory to C12-induced degradation of barrier function and activation of caspase 3/7, it seems unlikely that C12 activated apoptosis by increasing Ca cyto or that the antiapoptotic effects of Tg were mediated through an increase in Ca cyto .…”

“…The data are consistent with the idea that C12 has multiple, yet selective, effects on airway epithelial cells (e.g., C6 and C4 homoserine lactone have no effect on caspase 3/7 of airway epithelia). The inositol 1,4,5-trisphosphate receptor is activated, and Ca 2ϩ is released from the ER; Ca mito decreases, and mitochondria depolarize and release cytochrome c; proteins and oxidizing contents are released from the ER into the cytosol; nuclei shrink and fragment; and tight junctions degrade, and barrier function is lost (27,30, present data). Tg pretreatment inhibits most of these proapoptotic effects.…”

“…For measurements of transepithelial Cl Ϫ current (I Cl), Calu-3 cell monolayers were grown on Snapwell inserts, washed in PBS, mounted into water-jacketed (37°C) EasyMount Ussing chambers (Physiologic Instruments, San Diego, CA), and used for electrophysiological studies, as described previously (8,30). Transepithelial voltage (V T) was clamped to 0 mV, and RT and short-circuit current (I Cl under the conditions of these experiments) were measured using a four-electrode voltage clamp (VCC MC6 multichannel voltage clamp, Physiologic Instruments), with Ag-AgCl electrodes (World Precision Instruments, Sarasota, FL) connected to the solutions through agar bridges containing 1 M KCl.…”

“…A number of correlative data about Ca cyto , Ca mito , and Ca 2ϩ concentration in the ER (Ca er ) presented here (Fig. 8) and previously (4,27,30) may be informative about the mechanism. A summary of our results and speculations about C12 and Tg are presented in Fig.…”