Pseudomonas Aeruginosa Reduces Vx-809 Stimulated F508del-Cftr Chloride Secretion by Airway Epithelial Cells

Background: P. aeruginosa is an opportunistic pathogen that chronically infects the lungs of 85% of adult patients with Cystic Fibrosis (CF). Previously, we demonstrated that P. aeruginosa reduced wt-CFTR Cl secretion by airway epithelial cells. Recently, a new investigational drug VX-809 has been shown to increase F508del-CFTR Cl secretion in human bronchial epithelial (HBE) cells, and, in combination with VX-770, to increase FEV1 (forced expiratory volume in 1 second) by an average of 3-5% in CF patients homozygous for the F508del-CFTR mutation. We propose that P. aeruginosa infection of CF lungs reduces VX-809 + VX-770- stimulated F508del-CFTR Cl secretion, and thereby reduces the clinical efficacy of VX-809 + VX-770. Methods and Results: F508del-CFBE cells and primary cultures of CF-HBE cells (F508del/F508del) were exposed to VX-809 alone or a combination of VX-809 + VX-770 for 48 hours and the effect of P. aeruginosa on F508del-CFTR Cl secretion was measured in Ussing chambers. The effect of VX-809 on F508del-CFTR abundance was measured by cell surface biotinylation and western blot analysis. PAO1, PA14, PAK and 6 clinical isolates of P. aeruginosa (3 mucoid and 3 non-mucoid) significantly reduced drug stimulated F508del-CFTR Cl secretion, and plasma membrane F508del-CFTR. Conclusion: The observation that P. aeruginosa reduces VX-809 and VX-809 + VX-770 stimulated F508del CFTR Cl secretion may explain, in part, why VX-809 + VX-770 has modest efficacy in clinical trials

Farnesol Induces Hydrogen Peroxide Resistance in Candida albicans Yeast by Inhibiting the Ras-Cyclic AMP Signaling Pathway

Farnesol, a Candida albicans cell-cell signaling molecule that participates in the control of morphology, has an additional role in protection of the fungus against oxidative stress. In this report, we show that although farnesol induces the accumulation of intracellular reactive oxygen species (ROS), ROS generation is not necessary for the induction of catalase (Cat1)-mediated oxidative-stress resistance. Two antioxidants, α-tocopherol and, to a lesser extent, ascorbic acid effectively reduced intracellular ROS generation by farnesol but did not alter farnesol-induced oxidative-stress resistance. Farnesol inhibits the Ras1-adenylate cyclase (Cyr1) signaling pathway to achieve its effects on morphology under hypha-inducing conditions, and we demonstrate that farnesol induces oxidative-stress resistance by a similar mechanism. Strains lacking either Ras1 or Cyr1 no longer exhibited increased protection against hydrogen peroxide upon preincubation with farnesol. While we also observed the previously reported increase in the phosphorylation level of Hog1, a known regulator of oxidative-stress resistance, in the presence of farnesol, the hog1/hog1 mutant did not differ from wild-type strains in terms of farnesol-induced oxidative-stress resistance. Analysis of Hog1 levels and its phosphorylation states in different mutant backgrounds indicated that mutation of the components of the Ras1-adenylate cyclase pathway was sufficient to cause an increase of Hog1 phosphorylation even in the absence of farnesol or other exogenous sources of oxidative stress. This finding indicates the presence of unknown links between these signaling pathways. Our results suggest that farnesol effects on the Ras-adenylate cyclase cascade are responsible for many of the observed activities of this fungal signaling molecule

Farnesol and Cyclic AMP Signaling Effects on the Hypha-to-Yeast Transition in Candida Albicans

Candida albicans, a fungal pathogen of humans, regulates its morphology in response to many environmental cues and this morphological plasticity contributes to virulence. Farnesol, an autoregulatory molecule produced by C. albicans, inhibits the induction of hyphal growth by inhibiting adenylate cyclase (Cyr1). The role of farnesol and Cyr1 in controlling the maintenance of hyphal growth has been less clear. Here, we demonstrate that preformed hyphae transition to growth as yeast in response to farnesol and that strains with increased cyclic AMP (cAMP) signaling exhibit more resistance to farnesol. Exogenous farnesol did not induce the hypha-to-yeast transition in mutants lacking the Tup1 or Nrg1 transcriptional repressors in embedded conditions. Although body temperature is not required for embedded hyphal growth, we found that the effect of farnesol on the hypha-to-yeast transition varies inversely with temperature. Our model of Cyr1 activity being required for filamentation is also supported by our liquid assay data, which show increased yeast formation when preformed filaments are treated with farnesol. Together, these data suggest that farnesol can modulate morphology in preformed hyphal cells and that the repression of hyphal growth maintenance likely occurs through the inhibition of cAMP signaling

GbdR Regulates Pseudomonas aeruginosa plcH and pchP Transcription in Response to Choline Catabolites

Pseudomonas aeruginosa hemolytic phospholipase C, PlcH, can degrade phosphatidylcholine (PC) and sphingomyelin in eukaryotic cell membranes and extracellular PC in lung surfactant. Numerous studies implicate PlcH in P. aeruginosa virulence. The phosphorylcholine released by PlcH activity on phospholipids is hydrolyzed by a periplasmic phosphorylcholine phosphatase, PchP. Both plcH gene expression and PchP enzyme activity are positively regulated by phosphorylcholine degradation products, including glycine betaine. Here we report that the induction of plcH and pchP transcription by glycine betaine is mediated by GbdR, an AraC family transcription factor. Mutants that lack gbdR are unable to induce plcH and pchP in media containing glycine betaine or choline and in phosphatidylcholine-rich environments, such as lung surfactant or mouse lung lavage fluid. In T broth containing choline, the gbdR mutant exhibited a 95% reduction in PlcH activity. In electrophoretic mobility shift assays, a GbdR-maltose binding protein fusion bound specifically to both the plcH and pchP promoters. Promoter mapping, alignment of GbdR-regulated promoter sequences, and analysis of targeted promoter mutants that lack GbdR-dependent induction of transcription were used to identify a region necessary for GbdR-dependent transcriptional activation. GbdR also plays a significant role in plcH and pchP regulation within the mouse lung. Our studies suggest that GbdR is the primary regulator of plcH and pchP expression in PC-rich environments, such as the lung, and that pchP and other genes involved in phosphorylcholine catabolism are necessary to stimulate the GbdR-mediated positive feedback induction of plcH

Gene expression studies for the analysis of domoic acid production in the marine diatom Pseudo-nitzschia multiseries

Background: Pseudo-nitzschia multiseries Hasle (Hasle) (Ps-n) is distinctive among the ecologically important marine diatoms because it produces the neurotoxin domoic acid. Although the biology of Ps-n has been investigated intensely, the characterization of the genes and biochemical pathways leading to domoic acid biosynthesis has been limited. To identify transcripts whose levels correlate with domoic acid production, we analyzed Ps-n under conditions of high and low domoic acid production by cDNA microarray technology and reverse-transcription quantitative PCR (RT-qPCR) methods. Our goals included identifying and validating robust reference genes for Ps-n RNA expression analysis under these conditions. Results: Through microarray analysis of exponential- and stationary-phase cultures with low and high domoic acid production, respectively, we identified candidate reference genes whose transcripts did not vary across conditions. We tested eleven potential reference genes for stability using RT-qPCR and GeNorm analyses. Our results indicated that transcripts encoding JmjC, dynein, and histone H3 proteins were the most suitable for normalization of expression data under conditions of silicon-limitation, in late-exponential through stationary phase. The microarray studies identified a number of genes that were up- and down-regulated under toxin-producing conditions. RT-qPCR analysis, using the validated controls, confirmed the up-regulation of transcripts predicted to encode a cycloisomerase, an SLC6 transporter, phosphoenolpyruvate carboxykinase, glutamate dehydrogenase, a small heat shock protein, and an aldo-keto reductase, as well as the down-regulation of a transcript encoding a fucoxanthin-chlorophyll a-c binding protein, under these conditions. Conclusion: Our results provide a strong basis for further studies of RNA expression levels in Ps-n, which will contribute to our understanding of genes involved in the production and release of domoic acid, an important neurotoxin that affects human health as well as ecosystem function.Plymouth State University Graduate Programs OfficeWoods Hole Oceanographic Institution Academic Programs OfficeNew Hampshire IDeA Network of Biological Research Excellence (NH-INBRE)National Center for Research Resources (U.S.) (Grant 5P20RR030360-03)National Institute of General Medical Sciences (U.S.) (Grant 8P20GM103506-03

Using DTAGs to understand sound use, behavior, and vessel and associated noise effects in Southern Resident killer whales

Prey availability and disturbance from vessels and noise are identified threats to the recovery of endangered Southern Resident killer whales. Vessels and noise can mask echolocation signals used to capture fish prey and/or disrupt foraging behavior with implications for energy acquisition. In the U.S., vessel regulations have been implemented since 2011 to protect killer whales from vessel disturbance, particularly given the extent of whale-watching activities in the Salish Sea. We utilized suction cup-attached digital acoustic recording tags (DTAGs), consisting of hydrophones and movement sensors, to measure received noise levels, understanding killer whale use of sound, and determine effects of vessels and noise on subsurface behavior. During the 29 tag deployments on individually identified killer whales, we collected detailed geo-referenced vessel data concurrently as conditions allowed, along with opportunistic observations of predation to validate feeding. Received noise levels (dB re 1microPa) were significantly different across years but not consistently lower after the implementation of vessel regulations. Of the vessel factors considered, both vessel count and speed, but not distance, explained differences in noise levels, which may reflect changes in whale-watching vessel practices after regulations implementation. Additionally, the analysis of data from these animal-borne tags allow us to better understand subsurface foraging behavior involving the use of sound, to quantify foraging rates at an individual level, and to understand detailed vessel and noise effects. The results, along with those of other related studies, inform conservation and management measures that aim to promote Southern Resident recovery

Compendium-Wide Analysis of Pseudomonas aeruginosa Core and Accessory Genes Reveals Transcriptional Patterns across Strains PAO1 and PA14.

Pseudomonas aeruginosa is an opportunistic pathogen that causes difficult-to-treat infections. Two well-studied divergent P. aeruginosa strain types, PAO1 and PA14, have significant genomic heterogeneity, including diverse accessory genes present in only some strains. Genome content comparisons find core genes that are conserved across both PAO1 and PA14 strains and accessory genes that are present in only a subset of PAO1 and PA14 strains. Here, we use recently assembled transcriptome compendia of publicly available P. aeruginosa RNA sequencing (RNA-seq) samples to create two smaller compendia consisting of only strain PAO1 or strain PA14 samples with each aligned to their cognate reference genome. We confirmed strain annotations and identified other samples for inclusion by assessing each sample\u27s median expression of PAO1-only or PA14-only accessory genes. We then compared the patterns of core gene expression in each strain. To do so, we developed a method by which we analyzed genes in terms of which genes showed similar expression patterns across strain types. We found that some core genes had consistent correlated expression patterns across both compendia, while others were less stable in an interstrain comparison. For each accessory gene, we also determined core genes with correlated expression patterns. We found that stable core genes had fewer coexpressed neighbors that were accessory genes. Overall, this approach for analyzing expression patterns across strain types can be extended to other groups of genes, like phage genes, or applied for analyzing patterns beyond groups of strains, such as samples with different traits, to reveal a deeper understanding of regulation

The pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl&minus; ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in &alpha;/&beta; hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe. <br /

Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills Candida albicans in biofilms

Pseudomonas aeruginosa produces several phenazines including the recently described 5-methyl-phenazine-1-carboxylic acid (5MPCA), which exhibits a novel antibiotic activity towards pathogenic fungi such as Candida albicans . Here we characterize the unique antifungal mechanisms of 5MPCA using its analogue phenazine methosulphate (PMS). Like 5MPCA, PMS induced fungal red pigmentation and killing. Mass spectrometry analyses demonstrated that PMS can be covalently modified by amino acids, a process that yields red derivatives. Furthermore, soluble proteins from C. albicans grown with either PMS or P. aeruginosa were also red and demonstrated absorbance and fluorescence spectra similar to that of PMS covalently linked to either amino acids or proteins in vitro , suggesting that 5MPCA modification by protein amine groups occurs in vivo . The red-pigmented C. albicans soluble proteins were reduced by NADH and spontaneously oxidized by oxygen, a reaction that likely generates reactive oxygen species (ROS). Additional evidence indicated that ROS generation precedes 5MPCA-induced fungal death. Reducing conditions greatly enhanced PMS uptake by C. albicans and killing. Since 5MPCA was more toxic than other phenazines that are not modified, such as pyocyanin, we propose that the covalent binding of 5MPCA promotes its accumulation in target cells and contributes to its antifungal activity in mixed-species biofilms.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/1/j.1365-2958.2010.07414.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/79382/2/MMI_7414_sm_Figures_Table.pd