Pseudomonas aeruginosa uses quorum sensing, an interbacterial communication system, to regulate gene expression. The signaling molecule N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is thought to ...play a central role in quorum sensing. Since 3OC12-HSL can be degraded by paraoxonase (PON) family members, we hypothesized that PONs regulate P. aeruginosa virulence in vivo. We chose Drosophila melanogaster as our model organism because it has been shown to be a tractable model for investigating host-pathogen interactions and lacks PONs. By using quorum-sensing-deficient P. aeruginosa, synthetic acyl-HSLs, and transgenic expression of human PON1, we investigated the role of 3OC12-HSL and PON1 on P. aeruginosa virulence. We found that P. aeruginosa virulence in flies was dependent upon 3OC12-HSL. PON1 transgenic flies expressed enzymatically active PON1 and thereby exhibited arylesterase activity and resistance to organophosphate toxicity. Moreover, PON1 flies were protected from P. aeruginosa lethality, and protection was dependent on the lactonase activity of PON1. Our findings show that PON1 can interfere with quorum sensing in vivo and provide insight into what we believe is a novel role for PON1 in the innate immune response to quorum-sensing-dependent pathogens. These results raise intriguing possibilities about human-pathogen interactions, including potential roles for PON1 as a modifier gene and for PON1 protein as a regulator of normal bacterial florae, a link between infection/inflammation and cardiovascular disease, and a potential therapeutic modality.
Factors required for commitment of an undifferentiated airway epithelial cell to a ciliated cell are unknown. Cell ultrastructure analysis indicates ciliated cell commitment activates a multistage ...program involving synthesis of cilia precursor proteins and assembly of macromolecular complexes. Foxj1 is an f-box transcription factor expressed in ciliated cells and shown to be required for cilia formation by gene deletion in a mouse model. To identify a specific role for foxj1 in directing the ciliated cell phenotype, we evaluated the capacity of foxj1 to induce ciliogenesis and direct cilia assembly. In a primary culture model of wild-type mouse airway epithelial cells, foxj1 expression preceded the appearance of cilia and in cultured foxj1 null cells cilia did not develop. Delivery of foxj1 to polarized epithelial cell lines and primary cultured alveolar epithelial cells failed to promote ciliogenesis. Similarly, delivery of foxj1 to wild-type airway epithelial cells did not enhance the total number of ciliated cells. In contrast, delivery of foxj1 to null cells resulted in the appearance of cilia. Analysis revealed that, in the absence of foxj1, null cells contained cilia precursor basal bodies, indicating prior commitment to ciliogenesis. However, the basal bodies were disorganized within the apical compartment and failed to dock with the apical membrane. Reconstitution of foxj1 in null cells restored normal basal body organization, resulting in axoneme growth. Thus foxj1 functions in late-stage ciliogenesis to regulate programs promoting basal body docking and axoneme formation in cells previously committed to the ciliated cell phenotype.
In cystic fibrosis (CF), the loss of cystic fibrosis transmembrane conductance regulator (CFTR) mediated Cl− and HCO3− secretion across the epithelium acidifies the airway surface liquid (ASL). ...Acidic ASL alters two key host defense mechanisms: Rapid ASL bacterial killing and mucociliary transport (MCT). Aerosolized tromethamine (Tham) increases ASL pH and restores the ability of ASL to rapidly kill bacteria in CF pigs. In CF pigs, clearance of insufflated microdisks is interrupted due to abnormal mucus causing microdisks to abruptly recoil. Aerosolizing a reducing agent to break disulfide bonds that link mucins improves MCT. Here, we are interested in restoring MCT in CF by aerosolizing Tham, a buffer with a pH of 8.4. Because Tham is hypertonic to serum, we use an acidified formulation as a control. We measure MCT by tracking the caudal movement of individual tantalum microdisks with serial chest computed tomography scans. Alkaline Tham improves microdisk clearance to within the range of that seen in non‐CF pigs. It also partially reverses MCT defects, including reduced microdisk recoil and elapse time until they start moving after methacholine stimulation in CF pig airways. The effect is not due to hypertonicity, as it is not seen with acidified Tham or hypertonic saline. This finding indicates acidic ASL impairs CF MCT and suggests that alkalinization of ASL pH with inhaled Tham may improve CF airway disease.
Cystic fibrosis airways are acidic and as a result, submucosal gland secretions are abnormally elastic. Tromethamine, a tris buffer with long‐lasting effect on pH, alkalinizes the airways and reverses mucociliary clearance defects in CF.
Air pollution is a risk factor for respiratory infections, and one of its main components is particulate matter (PM), which is comprised of a number of particles that contain iron, such as coal fly ...ash (CFA). Since free iron concentrations are extremely low in airway surface liquid (ASL), we hypothesize that CFA impairs antimicrobial peptides (AMP) function and can be a source of iron to bacteria. We tested this hypothesis in vivo by instilling mice with Pseudomonas aeruginosa (PA01) and CFA and determine the percentage of bacterial clearance. In addition, we tested bacterial clearance in cell culture by exposing primary human airway epithelial cells to PA01 and CFA and determining the AMP activity and bacterial growth in vitro. We report that CFA is a bioavailable source of iron for bacteria. We show that CFA interferes with bacterial clearance in vivo and in primary human airway epithelial cultures. Also, we demonstrate that CFA inhibits AMP activity in vitro, which we propose as a mechanism of our cell culture and in vivo results. Furthermore, PA01 uses CFA as an iron source with a direct correlation between CFA iron dissolution and bacterial growth. CFA concentrations used are very relevant to human daily exposures, thus posing a potential public health risk for susceptible subjects. Although CFA provides a source of bioavailable iron for bacteria, not all CFA particles have the same biological effects, and their propensity for iron dissolution is an important factor. CFA impairs lung innate immune mechanisms of bacterial clearance, specifically AMP activity. We expect that identifying the PM mechanisms of respiratory infections will translate into public health policies aimed at controlling, not only concentration of PM exposure, but physicochemical characteristics that will potentially cause respiratory infections in susceptible individuals and populations.
Recombinant adeno-associated viruses (AAV) are promising gene therapy vectors. Whereas AAV serotype 2-mediated gene transfer to muscle has partially replaced factor IX deficiency in hemophilia ...patients, its ability to mediate gene transfer to the lungs for cystic fibrosis is hindered by lack of apical receptors. However, AAV serotype 5 infects human airway epithelia from the lumenal surface. We found that in contrast to AAV2, the apical membrane of airway epithelia contains abundant high affinity receptors for AAV5. Binding and gene transfer with AAV5 was abolished by genetic or enzymatic removal of sialic acid from the cell surface. Furthermore, binding and gene transfer to airway epithelia was competed by lectins that specifically bind 2,3-linked sialic acid. These observations suggest that 2,3-linked sialic acid is either a receptor for AAV5 or it is a necessary component of a receptor complex. Further elucidation of the receptor for this virus should enhance understanding of parvovirus biology and expand the therapeutic targets for AAV vectors.
Paraoxonases (PONs) are a family of lactonases with promiscuous enzyme activity that has been implicated in multiple diseases. PON2 is intracellularly located, is the most ubiquitously expressed PON, ...and has the highest lactonase activity of the PON family members. Whereas some single-nucleotide polymorphisms (SNPs) in PON1 have resulted in altered enzymatic activity in serum, to date the functional consequences of SNPs on PON2 function remain unknown. We hypothesized that a common PON2 SNP would result in impaired lactonase activity. Substitution of cysteine for serine at codon 311 in recombinant PON2 resulted in normal protein production and localization but altered glycosylation and decreased lactonase activity. Moreover, we screened 200 human lung samples for the PON2 Cys311 variant and found that in vivo this mutation impaired lactonase activity. These data suggest that impaired lactonase activity may play a role in innate immunity, atherosclerosis, and other diseases associated with the PON2 311 SNP.
Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule ...localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR(Ex7) and CAR(Ex8)). We found low-level expression of the CAR(Ex8) isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR(Ex7), CAR(Ex8) localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR(Ex7), CAR(Ex8) differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR(Ex8) providing a potential mechanism for the apical localization of CAR(Ex8) in airway epithelial. In summary, apical localization of CAR(Ex8) may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.
Paraoxonase 1 (PON1) is a protein found associated with high density lipoprotein (HDL), thought to prevent oxidative modification of low-density lipoprotein (LDL). This enzyme has been implicated in ...lowering the risk of cardiovascular disease. Anoxia-reoxygenation and oxidative stress are important elements in cardiovascular and cerebrovascular disease. However, the role of PON1 in anoxia-reoxygenation or anoxic injury is unclear. We hypothesize that PON1 prevents anoxia-reoxygenation injury. We set out to determine whether PON1 expression in Drosophila melanogaster protects against anoxia-reoxygenation (A-R) induced injury.
Wild type (WT) and transgenic PON1 flies were exposed to anoxia (100% Nitrogen) for different time intervals (from 1 to 24 hours). After the anoxic period, flies were placed in room air for reoxygenation. Activity and survival of flies was then recorded.
Within 5 minutes of anoxia, all flies fell into a stupor state. After reoxygenation, survivor flies resumed activity with some delay. Interestingly, transgenic flies recovered from stupor later than WT. PON1 transgenic flies had a significant survival advantage after A-R stress compared with WT. The protection conferred by PON1 expression was present regardless of the age or dietary restriction. Furthermore, PON1 expression exclusively in CNS conferred protection.
Our results support the hypothesis that PON1 has a protective role in anoxia-reoxygenation injury, and its expression in the CNS is sufficient and necessary to provide a 100% survival protection.
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