Viral-Bacterial Co-infections in the Cystic Fibrosis Respiratory Tract

A majority of the morbidity and mortality associated with the genetic disease Cystic Fibrosis (CF) is due to lung disease resulting from chronic respiratory infections. The CF airways become chronically colonized with bacteria in childhood, and over time commensal lung microbes are displaced by bacterial pathogens, leading to a decrease in microbial diversity that correlates with declining patient health. Infection with the pathogen Pseudomonas aeruginosa is a major predictor of morbidity and mortality in CF, with CF individuals often becoming chronically colonized with P. aeruginosa in early adulthood and thereafter having an increased risk of hospitalization. Progression of CF respiratory disease is also influenced by infection with respiratory viruses. Children and adults with CF experience frequent respiratory viral infections with respiratory syncytial virus (RSV), rhinovirus, influenza, parainfluenza, and adenovirus, with RSV and influenza infection linked to the greatest decreases in lung function. Along with directly causing severe respiratory symptoms in CF populations, the impact of respiratory virus infections may be more far-reaching, indirectly promoting bacterial persistence and pathogenesis in the CF respiratory tract. Acquisition of P. aeruginosa in CF patients correlates with seasonal respiratory virus infections, and CF patients colonized with P. aeruginosa experience increased severe exacerbations and declines in lung function during respiratory viral co-infection. In light of such observations, efforts to better understand the impact of viral-bacterial co-infections in the CF airways have been a focus of clinical and basic research in recent years. This review summarizes what has been learned about the interactions between viruses and bacteria in the CF upper and lower respiratory tract and how co-infections impact the health of individuals with CF

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 /

NADH Dehydrogenases in Pseudomonas aeruginosa Growth and Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen with a complex respiratory chain. The bacterium is predicted to express three NADH:ubiquinone oxidoreductases (NDH-1, NDH-2 and Nqr). We created deletions strains of the predicted NADH:ubiquinone oxidoreductases alone, and in combination to determine the respective roles of the NADH dehydrogenases in growth and virulence. NDH-1 and NDH-2 were largely redundant under aerobic conditions. Aerobic NADH dehydrogenase enzymatic activity assay was lost with deletion of both NDH-1 and NDH-2. Under anaerobic conditions, NDH-1 was required for robust growth, and overexpression of NDH-2 rescued the NDH-1 anaerobic growth defect in rich media. There was not compensatory upregulation of NDH-2 under anaerobic conditions in NDH-1 deletion strains. To test which genes were required for in vivo virulence, we used both an insect and plant disease model. In the Galleria mellonella model, neither deletion of NDH-1 nor NDH-2 led to a change in median lethal dose, although death occurred more slowly in the NDH-1 deletion infections. In a lettuce model of virulence, loss of NDH-1 caused a decrease in recovered viable bacteria and a decrease in visual tissue damage. The compound deletion of NDH-1/NDH-2 causes a severe growth defect, both under aerobic and anaerobic conditions, and was avirulent in a lettuce model. Together, these results demonstrate the redundancy of the P. aeruginosa respiratory chain at the NADH dehydrogenase level in aerobic growth and virulence

Serum and glucocorticoid-inducible kinase1 increases plasma membrane wt-CFTR in human airway epithelial cells by inhibiting its endocytic retrieval

Background: Chloride (Cl) secretion by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) located in the apical membrane of respiratory epithelial cells plays a critical role in maintenance of the airway surface liquid and mucociliary clearance of pathogens. Previously, we and others have shown that the serum and glucocorticoid-inducible kinase-1 (SGK1) increases wild type CFTR (wt-CFTR) mediated Cl transport in Xenopus oocytes by increasing the amount of wt-CFTR protein in the plasma membrane. However, the effect of SGK1 on the membrane abundance of wt-CFTR in airway epithelial cells has not been examined, and the mechanism whereby SGK1 increases membrane wt-CFTR has also not been examined. Thus, the goal of this study was to elucidate the mechanism whereby SGK1 regulates the membrane abundance of wt-CFTR in human airway epithelial cells. Methods and Results: We report that elevated levels of SGK1, induced by dexamethasone, increase plasma membrane abundance of wt-CFTR. Reduction of SGK1 expression by siRNA (siSGK1) and inhibition of SGK1 activity by the SGK inhibitor GSK 650394 abrogated the ability of dexamethasone to increase plasma membrane wt-CFTR. Overexpression of a constitutively active SGK1 (SGK1-S422D) increased plasma membrane abundance of wt-CFTR. To understand the mechanism whereby SGK1 increased plasma membrane wt-CFTR, we examined the effects of siSGK1 and SGK1-S442D on the endocytic retrieval of wt-CFTR. While siSGK1 increased wt-CFTR endocytosis, SGK1-S442D inhibited CFTR endocytosis. Neither siSGK1 nor SGK1-S442D altered the recycling of endocytosed wt-CFTR back to the plasma membrane. By contrast, SGK1 increased the endocytosis of the epidermal growth factor receptor (EGFR). Conclusion: This study demonstrates for the first time that SGK1 selectively increases wt-CFTR in the plasma membrane of human airway epithelia cells by inhibiting its endocytic retrieval from the membrane. © 2014 Bomberger et al

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions

A recent workshop titled “Developing Models to Study Polymicrobial Infections,” sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 351 investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally

A genome-wide association study of severe asthma exacerbations in Latino children and adolescents

Severe asthma exacerbations are a major cause of school absences and healthcare costs in children, particularly those in high-risk racial/ethnic groups. To identify susceptibility genes for severe asthma exacerbations in Latino children and adolescents, we conducted a meta-analysis of genome-wide association studies (GWAS) in 4010 Latino youth with asthma in four independent cohorts, including 1693 Puerto Ricans, 1019 Costa Ricans, 640 Mexicans, 256 Brazilians, and 402 members of other Latino subgroups. We then conducted methylation quantitative trait locus (mQTL), expression quantitative trait locus (eQTL), and expression quantitative trait methylation (eQTM) analyses to assess whether the top SNP in the meta-analysis is linked to DNA methylation and gene expression in nasal (airway) epithelium in separate cohorts of Puerto Rican and Dutch children and adolescents. In the meta-analysis of GWAS, a SNP in FLJ22447 (rs2253681) was significantly associated with 1.55 increased odds of severe asthma exacerbations (95% confidence interval=1.34 to 1.79, p=6.3×10-9). This SNP was significantly associated with DNA methylation of a CpG site (cg25024579) at the FLJ22447 locus, which was in turn associated with increased expression of KCNJ2-AS1 in nasal airway epithelium from Puerto Rican children and adolescents (β=0.10, p=2.18×10-7). Thus, SNP rs2253681 was significantly associated with both DNA methylation of a cis-CpG in FLJ22447 and severe asthma exacerbations in Latino youth. This may be partly explained by changes in airway epithelial expression of a gene recently implicated in atopic asthma in Puerto Rican children and adolescents (KCNJ2-AS1)

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

Training wildlife biologists for work on private lands

Moderator: John Calderazzo.Presented at the 8th international congress for wildlife and livelihoods on private and communal lands: livestock, tourism, and spirit, that was held on September 7-12, 2014 in Estes Park, Colorado.Video presenter: Jennifer Smith.Conservation and management of wildlife on private lands in the United States is a critical component of the wildlife profession, although university curricula focus on public lands management. Wildlife biologists working on private lands are faced with a mutable landscape and pressures of alternative land uses that shift with changes in economics, regulatory regimes, and societal or cultural values. Loss of biodiversity can be lessened if wildlife biologists are better prepared to work in this challenging landscape. Colleges and universities are faced with training wildlife biologists to work on both public and private lands. Unfortunately, however, wildlife training programs often fail to integrate the management techniques, policy, economics, human dimensions, problem solving, and communication skills required by private lands professionals. Of 14 leading undergraduate wildlife programs that we identified, 36% required a policy class, 36% required a human dimensions class and 7% required an economics class; although most universities did offer interdisciplinary courses, participation was seldom mandatory. We suggest that students and current professionals need to be trained in the skills necessary to protect wildlife when working on privately owned lands. We highlight tools needed for effective private lands conservation such as economic incentives, education, and outreach, and we suggest ways in which they can be taught through modifications to current curriculum, short-courses, continuing education credits, certificate programs, internships, externships, and involvement of agency personnel in the classroom

Model of increased susceptibility to secondary bacterial infection after primary viral infection of the respiratory epithelium.

<p>(A) The respiratory epithelium restricts bacterial attachment via mucociliary clearance and maintenance of cell–cell junctions, which restricts access to bacterial receptors. During viral infection, ciliary beat is reduced, barrier function is disrupted, bacterial receptors (purple) are upregulated, and direct viral–bacterial interactions lead to increased bacterial adherence to the epithelium. (B) The respiratory epithelium recruits and activates neutrophils or polymorphonuclear (PMN, red) cells and T helper cells (particularly Th17, green) in response to detection of bacterial infection or pro-inflammatory cytokines. These effects lead to influx of neutrophils and stimulation of epithelial antimicrobial peptide/protein (AMP, orange spirals) production in response to IL-17 receptor signaling (green circles, dashed arrow) by the epithelium. During viral infection, epithelial cells produce interferons (IFN), which skew the immune status towards antiviral activity, suppressing neutrophil, Th17 responses, and other antibacterial functions. (C) To inhibit microbial growth, the respiratory epithelium actively restricts nutrient availability in the airway lumen, including limitation of luminal iron concentrations (Fe, red circles). During viral infection, interferon production leads to dysfunctional iron limitation, stimulating biofilm biogenesis. In the case of influenza infection, influenza stimulates mucin secretion and cleaves sialic acid (purple hexagons) from secreted mucins via neuraminidase activity. Common upper respiratory tract commensal bacteria can utilize liberated sialic acid as a nutrient.</p