Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a significant contributor to recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and to promote biofilm formation. Quorum sensing (QS) is a mechanism wherein P. aeruginosa secretes small diffusible molecules, specifically acyl homo serine lactones, such as N-(3-oxo-dodecanoyl)-l-homoserine lactone (3O-C12-HSL), that promote biofilm formation and virulence via interbacterial communication. Strategies that strengthen the host's ability to inhibit bacterial virulence would enhance host defenses and improve the treatment of resistant infections. We have recently shown that peroxisome proliferator-activated receptor γ (PPARγ) agonists are potent immunostimulators that play a pivotal role in host response to virulent P. aeruginosa Here, we show that QS genes in P. aeruginosa (strain PAO1) and 3O-C12-HSL attenuate PPARγ expression in bronchial epithelial cells. PAO1 and 3O-C12-HSL induce barrier derangements in bronchial epithelial cells by lowering the expression of junctional proteins, such as zonula occludens-1, occludin, and claudin-4. Expression of these proteins was restored in cells that were treated with pioglitazone, a PPARγ agonist, before infection with PAO1 and 3O-C12-HSL. Barrier function and bacterial permeation studies that have been performed in primary human epithelial cells showed that PPARγ agonists are able to restore barrier integrity and function that are disrupted by PAO1 and 3O-C12-HSL. Mechanistically, we show that these effects are dependent on the induction of paraoxonase-2, a QS hydrolyzing enzyme, that mitigates the effects of QS molecules. Importantly, our data show that pioglitazone, a PPARγ agonist, significantly inhibits biofilm formation on epithelial cells by a mechanism that is mediated via paraoxonase-2. These findings elucidate a novel role for PPARγ in host defense against P. aeruginosa Strategies that activate PPARγ can provide a therapeutic complement for treatment of resistant P. aeruginosa infections.-Bedi, B., Maurice, N. M., Ciavatta, V. T., Lynn, K. S., Yuan, Z., Molina, S. A., Joo, M., Tyor, W. R., Goldberg, J. B., Koval, M., Hart, C. M., Sadikot, R. T. Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease.

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease.

Emission, Raman spectroscopy, and structural characterization of actinide tetracyanometallates.

Three new compounds, {U2(H2O)10(O)[Pt(CN)4]3}·4H2O, {Th2(H2O)10(OH)2[Pd(CN)4]3}·8H2O, and {(UO2)2(DMSO)4(OH)2[Ni(CN)4]}, in the actinide tetracyanometallate, Anx[M(CN)4]y, class of compounds have been synthesized and characterized by confocal Raman spectroscopy and single crystal X-ray diffraction. These compounds contain unique structures illustrating dimeric actinide species. The absence of intense charge transfer emission in the visible range for {U2(H2O)10(O)[Pt(CN)4]3}·4H2O, as compared to the platinum starting material, is unusual because of the presence of pseudo-one-dimensional Pt···Pt chains in this compound. Confocal Raman spectroscopy of the cyanide stretching region provides insight into the binding domain (mono-, bi-, tri-, tetradentate) of the tetracyanometallates in these novel structures.

Asymmetric distribution of dynamin-2 and ß-catenin relative to tight junction spikes in alveolar epithelial cells.

Tight junctions between lung alveolar epithelial cells maintain an air-liquid barrier necessary for healthy lung function. Previously, we found that rearrangement of tight junctions from a linear, cortical orientation into perpendicular protrusions (tight junction spikes) is associated with a decrease in alveolar barrier function, especially in alcoholic lung syndrome. Using quantitative super-resolution microscopy, we found that spikes in control cells were enriched for claudin-18 as compared with alcohol-exposed cells. Moreover, using an in situ method to measure barrier function, tight junction spikes were not associated with localized increases in permeability. This suggests that tight junction spikes have a regulatory role as opposed to causing a physical weakening of the epithelial barrier. We found that tight junction spikes form at cell-cell junctions oriented away from pools of ß-catenin associated with actin filaments, suggesting that adherens junctions determine the directionality of tight junction spikes. Dynamin-2 was associated with junctional claudin-18 and ZO-1, but showed little localization with ß-catenin and tight junction spikes. Treatment with Dynasore decreased the number of tight junction spikes/cell, increased tight junction spike length, and stimulated actin to redistribute to cortical tight junctions. By contrast, Dynole 34-2 and MiTMAB altered ß-catenin localization, and reduced tight junction spike length. These data suggest a novel role for dynamin-2 in tight junction spike formation by reorienting junction-associated actin. Moreover, the greater spatial separation of adherens and tight junctions in squamous alveolar epithelial cells as compared with columnar epithelial cells facilitates analysis of molecular regulation of the apical junctional complex.

Ruffles and spikes: Control of tight junction morphology and permeability by claudins.

Epithelial barrier function is regulated by a family of transmembrane proteins known as claudins. Functional tight junctions are formed when claudins interact with other transmembrane proteins, cytosolic scaffold proteins and the actin cytoskeleton. The predominant scaffold protein, zonula occludens-1 (ZO-1), directly binds to most claudin C-terminal domains, crosslinking them to the actin cytoskeleton. When imaged by immunofluorescence microscopy, tight junctions most frequently are linear structures that form between tricellular junctions. However, tight junctions also adapt non-linear architectures exhibiting either a ruffled or spiked morphology, which both are responses to changes in claudin engagement of actin filaments. Other terms for ruffled tight junctions include wavy, tortuous, undulating, serpentine or zig-zag junctions. Ruffling is under the control of hypoxia induced factor (HIF) and integrin-mediated signaling, as well as direct mechanical stimulation. Tight junction ruffling is specifically enhanced by claudin-2, antagonized by claudin-1 and requires claudin binding to ZO-1. Tight junction spikes are sites of active vesicle budding and fusion that appear as perpendicular projections oriented towards the nucleus. Spikes share molecular features with focal adherens junctions and tubulobulbar complexes found in Sertoli cells. Lung epithelial cells under stress form spikes due to an increase in claudin-5 expression that directly disrupts claudin-18/ZO-1 interactions. Together this suggests that claudins are not simply passive cargoes controlled by scaffold proteins. We propose a model where claudins specifically influence tight junction scaffold proteins to control interactions with the cytoskeleton as a mechanism that regulates tight junction assembly and function.

Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions.

Claudins are tetraspan transmembrane tight-junction proteins that regulate epithelial barriers. In the distal airspaces of the lung, alveolar epithelial tight junctions are crucial to regulate airspace fluid. Chronic alcohol abuse weakens alveolar tight junctions, priming the lung for acute respiratory distress syndrome, a frequently lethal condition caused by airspace flooding. Here we demonstrate that in response to alcohol, increased claudin-5 paradoxically accompanies an increase in paracellular leak and rearrangement of alveolar tight junctions. Claudin-5 is necessary and sufficient to diminish alveolar epithelial barrier function by impairing the ability of claudin-18 to interact with a scaffold protein, zonula occludens 1 (ZO-1), demonstrating that one claudin affects the ability of another claudin to interact with the tight-junction scaffold. Critically, a claudin-5 peptide mimetic reverses the deleterious effects of alcohol on alveolar barrier function. Thus, claudin controlled claudin-scaffold protein interactions are a novel target to regulate tight-junction permeability.