Bonita Coutermarsh

Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

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 Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in individuals with cystic fibrosis, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl- channels in the apical plasma membrane. Rescue of ΔF508-CFTR by reduced temperature or chemical means reveals that the ΔF508 mutation reduces the half-life of ΔF508-CFTR in the apical plasma membrane. Because ΔF508-CFTR retains some Cl- channel activity, increased expression of ΔF508-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 ΔF508-CFTR in polarized human airway epithelial cells. Accordingly, the goal of this study was to determine the cellular defects in the trafficking of rescued ΔF508-CFTR that lead to the decreased apical membrane half-life of ΔF508-CFTR in polarized human airway epithelial cells. We report that in polarized human airway epithelial cells (CFBE41o-) the ΔF508 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.

Myosin Vb Is Required for Trafficking of the Cystic Fibrosis Transmembrane Conductance Regulator in Rab11a-specific Apical Recycling Endosomes in Polarized Human Airway Epithelial Cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl- channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and ΔF508-CFTR in the apical membrane and decreased CFTR-mediated Cl- secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated Cl- channel expressed in the apical plasma membrane in fluid-transporting epithelia. Although CFTR is rapidly endocytosed from the apical membrane of polarized epithelial cells and efficiently recycled back to the plasma membrane, little is known about the molecular mechanisms regulating CFTR endocytosis and endocytic recycling. Myosin VI, an actin-dependent, minus-end directed mechanoenzyme, has been implicated in clathrin-mediated endocytosis in epithelial cells. The goal of this study was to determine whether myosin VI regulates CFTR endocytosis. Endogenous, apical membrane CFTR in polarized human airway epithelial cells (Calu-3) formed a complex with myosin VI, the myosin VI adaptor protein Disabled 2 (Dab2), and clathrin. The tail domain of myosin VI, a dominant-negative recombinant fragment, displaced endogenous myosin VI from interacting with Dab2 and CFTR and increased the expression of CFTR in the plasma membrane by reducing CFTR endocytosis. However, the myosin VI tail fragment had no effect on the recycling of endocytosed CFTR or on fluid-phase endocytosis. CFTR endocytosis was decreased by cytochalasin D, an actin-filament depolymerizing agent. Taken together, these data indicate that myosin VI and Dab2 facilitate CFTR endocytosis by a mechanism that requires actin filaments.

IMMUNOMAGNETIC SEPARATION, PRIMARY CULTURE, AND CHARACTERIZATION OF CORTICAL THICK ASCENDING LIMB PLUS DISTAL CONVOLUTED TUBULE CELLS FROM MOUSE KIDNEY

SummaryRenal cortical thick ascending limbs of Henle’s loop (CAL) and distal convoluted tubules (DCT) represent sites at which much of the final regulation of urinary ionic composition, particularly that of calcium, is accomplished in both humans and in rodents. We sought in the present work to develop an efficient means for isolating parathyroid hormone (PTH)-sensitive cells from these nephron segments and to grow them in primary culture. [CAL+DCT] cells were isolated from mouse kidney using an antiserum against the Tamm-Horsfall glycoprotein which, in the renal cortex, is produced exclusively by these cells. A second antibody conjugated to coated ferrous particles permitted magnetic separation of [CAL+DCT] cells from Tamm-Horsfall negative renal cortical cells. Approximately 3 × 106 cells per kidney with a trypan blue exclusion greater than 94% were isolated by these procedures. Experiments were performed to characterize the cells after 7 to 10 days in primary culture. PTH and isoproterenol, but neither calcitonin nor vasopressin, stimulated cyclic AMP (cAMP) formation in [CAL+DCT] cells, consistent with the pattern of hormone-activated cAMP synthesis found in freshly isolated CAL and DCT segments. Alkaline phosphatase, an enzyme present dominantly in proximal tubule brush border membranes, was virtually absent from [CAL+DCT] cells but was present in Tamm-Horsfall negative cells. Similarly, Na-glucose cotransport was absent in [CAL+DCT] cells but present in Tamm-Horsfall negative renal cortical cells. Finally, transport-related oxygen consumption in [CAL+DCT] cells was blocked by bumetanide and by chlorothiazide, diuretics that inhibit sodium transport in CAL and DCT nephron segments. These results demonstrate that PTH-sensitive [CAL+DCT] cells can be isolated in relatively high yield and viability and grown in cell culture. Primary cultures of these cells exhibit a phenotype appropriate to their site of origin in the nephron.

Targeting CAL as a Negative Regulator of ΔF508-CFTR Cell-Surface Expression AN RNA INTERFERENCE AND STRUCTURE-BASED MUTAGENETIC APPROACH

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ΔF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL·CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

The Role of SGK and CFTR in Acute Adaptation to Seawater in Fundulus Heteroclitus

Killifish are euryhaline teleosts that adapt to increased salinity by up regulating CFTR mediated Cl- secretion in the gill and opercular membrane. Although many studies have examined the mechanisms responsible for long term (days) adaptation to increased salinity, little is known about the mechanisms responsible for acute (hours) adaptation. Thus, studies were conducted to test the hypotheses that the acute homeostatic regulation of NaCl balance in killifish involves a translocation of CFTR to the plasma membrane and that this effect is mediated by serum-and glucocorticoid-inducible kinase (SGK1). Cell surface biotinyation and Ussing chamber studies revealed that freshwater to seawater transfer rapidly (1 hour) increased CFTR Cl- secretion and the abundance of CFTR in the plasma membrane of opercular membranes. Q-RT-PCR and Western blot studies demonstrated that the increase in plasma membrane CFTR was preceded by an increase in SGK1 mRNA and protein levels. Seawater rapidly (1 hr) increases cortisol and plasma tonicity, potent stimuli of SGK1 expression, yet RU486, a glucocorticoid receptor antagonist, did not block the increase in SGK1 expression. Thus, in killifish SGK1 does not appear to be regulated by the glucocorticoid receptor. Since SGK1 has been shown to increase the plasma membrane abundance of CFTR in Xenopus oocytes, these observations suggest that acute adaptation (hours) to increased salinity in killifish involves translocation of CFTR from an intracellular pool to the plasma membrane, and that this effect may be mediated by SGK1.

Na+-Phosphate Cotransport in Mouse Distal Convoluted Tubule Cells: Evidence for Glvr-1 and Ram-1 Gene Expression

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+‐dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+‐dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 ± 3% of control), a competitive inhibitor of Na+‐Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi‐free medium for 21 h increased Na+‐Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12‐myristate 13‐acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+‐Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+‐Pi cotransport, suggesting that other Na+‐Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr‐1) and amphoteric murine retrovirus (Ram‐1) that also function as Na+‐Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+‐Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+‐Pi cotransport in the proximal and distal segments of the nephron.

Regulation of Renal Parathyroid Hormone Receptor Expression by 1,25-Dihydroxyvitamin D3 and Retinoic Acid

The renal distal convoluted tubule (DCT) is the major site of parathyroid hormone (PTH) and 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]-regulated calcium absorption. 1,25(OH)₂D₃ augments PTH-stimulated calcium transport by DCT cells, while having no effect of its own. 1,25(OH)₂D₃ mediates its effects on gene expression by binding to a nuclear vitamin-D receptor (VDR), which then associates with the retinoid-X receptor (RXR) as a heterodimer. We studied the effects of 1,25(OH)₂D₃, 9-cis- and all-trans-retinoic acid on PTH/PTHrP receptor expression. mRNAs for the PTH/PTHrP, VDR, and RXR receptors were detected in immortalized DCT cells by reverse transcriptase-polymerase chain reaction. Changes in PTH/PTHrP receptor mRNA expression were quantified by slot blot hybridization. 1,25(OH)₂D₃ maximally increased PTH/PTHrP receptor mRNA levels by 70%. The stimulation was specific since 1,25(OH)₂D₃ treatment had no effect on the expression of adrenergic receptor or Na⁺/H⁺ exchanger mRNA levels. Likewise, the inactive form, 25(OH)₂D₃ had no effect on PTH/PTHrP receptor mRNA expression. In combination with the putative RXR ligand, 9-cis-retinoic acid, 1,25(OH)₂D₃ increased PTH/PTHrP receptor mRNA levels 4-fold. 9-cis-Retinoic acid had no effect of its own on steady-state PTH/PTHrP receptor mRNA expression. The putative ligand for the retinoic acid receptor, all-trans-retinoic acid, increased PTH/PTHrP receptor mRNA expression alone and in combination with 1,25(OH)₂D₃. 9-cis-Retinoic acid alone, and in combination with 1,25(OH)₂D₃, also increased specific PTH/PTHrP receptor binding to plasma membranes isolated from DCT cells. These results indicate that 1,25(OH)₂D₃ upregulated PTH/PTHrP receptor expression at both mRNA and protein levels in a manner consistent with VDR/RXR heterodimers transactivating the PTH/PTHrP receptor gene by binding a vitamin D response element in the PTH/PTHrP gene.

Arsenic promotes Ubiquitinylation and lysosomal degradation of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels in human airway epithelial cells

Background: Arsenic increases respiratory bacterial infections by an unknown mechanism. Results: Arsenic increased the c-Cbl-mediated ubiquitinylation and degradation of CFTR in human lung cells and reduced CFTR chloride secretion. Conclusion: The reduction in chloride secretion is proposed to decrease mucociliary clearance of respiratory pathogens. Significance: Low levels of arsenic commonly found in food and water suppress the innate immune function of the lung. Arsenic exposure significantly increases respiratory bacterial infections and reduces the ability of the innate immune system to eliminate bacterial infections. Recently, we observed in the gill of killifish, an environmental model organism, that arsenic exposure induced the ubiquitinylation and degradation of cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that is essential for the mucociliary clearance of respiratory pathogens in humans. Accordingly, in this study, we tested the hypothesis that low dose arsenic exposure reduces the abundance and function of CFTR in human airway epithelial cells. Arsenic induced a time- and dose-dependent increase in multiubiquitinylated CFTR, which led to its lysosomal degradation, and a decrease in CFTR-mediated chloride secretion. Although arsenic had no effect on the abundance or activity of USP10, a deubiquitinylating enzyme, siRNA-mediated knockdown of c-Cbl, an E3 ubiquitin ligase, abolished the arsenic-stimulated degradation of CFTR. Arsenic enhanced the degradation of CFTR by increasing phosphorylated c-Cbl, which increased its interaction with CFTR, and subsequent ubiquitinylation of CFTR. Because epidemiological studies have shown that arsenic increases the incidence of respiratory infections, this study suggests that one potential mechanism of this effect involves arsenic-induced ubiquitinylation and degradation of CFTR, which decreases chloride secretion and airway surface liquid volume, effects that would be proposed to reduce mucociliary clearance of respiratory pathogens.