Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner.

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ⌬F508, causes retention of ⌬F508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl ؊ channels in the apical plasma membrane. Rescue of ⌬F508-CFTR by reduced temperature or chemical means reveals that the ⌬F508 mutation reduces the half-life of ⌬F508-CFTR in the apical plasma membrane. Because ⌬F508-CFTR retains some Cl ؊ channel activity, increased expression of ⌬F508-CFTR in the apical membrane could serve as a potential therapeutic approach for cystic fibrosis. However, little is known about the mechanisms responsible for the short apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ⌬F508-CFTR that lead to the decreased apical membrane half-life of ⌬F508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o؊) the ⌬F508 mutation increased endocytosis of CFTR from the apical membrane without causing a global endocytic defect or affecting the endocytic recycling of CFTR in the Rab11a-specific apical recycling compartment.The cystic fibrosis transmembrane conductance regulator (CFTR) 2 is an ATP binding cassette (ABC) transporter and a cAMP-regulated Cl Ϫ channel that mediates transepithelial Cl Ϫ transport in the airways, intestine, pancreas, testis, and other tissues (1-3). Cystic fibrosis (CF), a lethal genetic disease, is caused by mutations in the CFTR gene (1, 2). The most common mutation in CFTR is ⌬F508 (4, 5). ⌬F508-CFTR does not fold properly, and most of the protein is retained within the endoplasmic reticulum (ER) where it is subsequently degraded (5, 6). Several studies suggest that the ER retention of ⌬F508-CFTR is not complete, and some ⌬F508-CFTR is constitutively expressed in the plasma membrane of primary epithelial cells from individuals homozygous for the ⌬F508 mutation (7-10). Because ⌬F508-CFTR retains some Cl Ϫ channel activity when expressed in the plasma membrane (5,6,(11)(12)(13)(14), it would be desirable to increase the expression of ⌬F508-CFTR in the plasma membrane to alleviate the symptoms in CF patients. The trafficking of ⌬F508-CFTR to the plasma membrane can be increased by chemical means or reduced temperature (15-21). Yet, functional and biochemical studies in heterologous cell lines demonstrate that rescued ⌬F508-CFTR has a greatly reduced stability or halflife in the post-ER compartments, including the plasma membrane (13,(22)(23)(24). Very little is known about the apical membrane half-life of rescued ⌬F508-CFTR in polarized human airway epithelial cells. A recent study demonstrates that the functional stability of ⌬F508-CFTR in the apical membrane of differentiated respiratory epithelial cells derived from nasal polyps from individuals homozygous for the ⌬F508 mutation is decreased compared with WT-CFTR (25). Furthermore, the bioc...

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-⌬TRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-⌬TRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from ϳ24 to ϳ13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-⌬TRL did not differ. However, endocytic recycling of CFTR-⌬TRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.

Nephropathy is a major contributor to overall morbidity and mortality in diabetic patients. Early renal changes during diabetes include Na retention and renal hypertrophy. Tumor necrosis factor (TNF) is elevated during diabetes and is implicated in the pathogenesis of diabetic nephropathy. We tested the hypothesis that TNF contributes to Na retention and renal hypertrophy during diabetes. Rats with streptozotocin-induced diabetes exhibit increased urinary TNF excretion, Na retention, and renal hypertrophy through the first 20 days of diabetes. Administration of a soluble TNF antagonist (TNFR:Fc) to diabetic rats reduces urinary TNF excretion and prevents Na retention and renal hypertrophy. TNF stimulates Na uptake in distal tubule cells isolated from diabetic rats, providing a possible mechanism for TNF-induced Na retention. We conclude that urinary TNF contributes to early diabetic nephropathy and may serve as a valuable diagnostic marker. Furthermore, inhibition of TNF during diabetes may attenuate early pathological changes in diabetic nephropathy.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl؊ secretion across fluid-transporting epithelia is regu-