Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs

Pseudomonas aeruginosa chronically colonizing the lungs of cystic fibrosis (CF) patients undergoes fast evolution leading to clonal divergence. More than half of the genotypes of P. aeruginosa clone C isolates exclusively from CF lung infection exhibit large chromosomal inversions (LCIs). To analyse the impact of LCIs, as a novel mechanism of bacterial adaptation, the underlying molecular mechanism was examined. Analysis of inversion breakpoints suggested an IS6100-induced coupled insertion-inversion mechanism. A selective advantage was created by insertion of IS6100 into wbpM, pilB and mutS which leads to common CF phenotypes such as O-antigen and type IV pili deficiency and hypermutability. Speciation in bacteria is accompanied by LCIs. Therefore adaptation by LCIs that allows persistence of P. aeruginosa in the CF lung and species diversification in that new ecological niche can serve as a model for bacterial genome evolution.     

Regulation of matrilysin expression in airway epithelial cells by Pseudomonas aeruginosa flagellin

Matrilysin (matrix metalloproteinase-7) is expressed by mucosal epithelia throughout the body and functions in host defense by activating murine intestinal alpha-defensins. In normal adult human lung, matrilysin is expressed at low levels in the airway epithelium, but is markedly up-regulated in cystic fibrosis (CF). Because CF lungs support a heavy bacterial load, we assessed if relevant CF pathogens regulate matrilysin expression in human lung epithelial cells. Indeed, acute infection with Pseudomonas aeruginosa (but not Staphylococcus aureus, Haemophilus influenzae, or Klebsiella pneumoniae) induced the expression of matrilysin in Calu-3 lung epithelial cells. Increased matrilysin mRNA levels were detectable at 3 h post-infection and peaked at a 25-fold induction between 6 and 8 h. Both P. aeruginosa CF isolates and laboratory strains induced matrilysin expression to similar levels. Flagellin, the monomeric precursor of bacterial flagella, was identified as the inductive factor released by P. aeruginosa that regulated matrilysin expression. In addition, flagellin-null mutants failed to stimulate matrilysin expression in cultured cells or in lungs infected in vivo. These data show that P. aeruginosa (and specifically flagellin) potently stimulates matrilysin expression in lung epithelial cells and may mediate the overexpression of this proteinase in CF lungs.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in Cftr knockout mice using novel non-invasive direct pulmonary infection technique

To better understand the mechanism of lung infection with Pseudomonas aeruginosa (P. aeruginosa), many techniques have been developed in order to establish lung infection in rodents. A model of chronic lung infection, using tracheotomy to inoculate the bacteria, has been extensively used in the cystic fibrosis (CF) mouse model of lung infection. The cystic fibrosis transmembrane channel (Cftr) knockout (KO) mice are smaller than normal mice and are more sensitive to housing and nutritional conditions, leading to small amounts of animals being available for experiments. Because of these characteristics, and because of the invasiveness of the infection procedure which we, and others, have been using to mimic the lung infection, we sought to find an alternative way to study the inflammatory response during lung P. aeruginosa infection. The technique we describe here consists of the injection of bacterial beads directly into the lungs through the mouth without the need of any tracheal incisions. This technique of direct pulmonary delivery enables much faster infection of the animals compared with the intratracheal technique previously used. The use of this less invasive technique allows the exclusion of the surgery-related inflammation. Our results show that, using the direct pulmonary delivery technique, the KO mice were more susceptible to P. aeruginosa lung infection compared with their wild-type (WT) controls, as shown by their increased weight loss, higher bacterial burden and more elevated polymorphonuclear (PMN) alveolar cell recruitment into the lungs. These differences are consistent with the pathological profiles observed in CF patients infected with P. aeruginosa. Overall, this method simplifies the infection procedure in terms of its duration and invasiveness, and improves the survival rate of the KO mice when compared with the previously used intratracheal procedure.     

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

Inflammatory markers in cystic fibrosis patients with lung Pseudomonas aeruginosa infection

Chronic endobronchial inflammation and bacterial infection are the main causes of morbidity and mortality in cystic fibrosis (CF), an autosomal recessive genetic disorder associated with improper function of chloride channels. Inflammation in CF lung is greatly amplified after Pseudomonas aeruginosa infection. In this study the relationship between P. aeruginosa status and inflammatory markers has been investigated. Seventeen CF children in acute lung exacerbation were examined. CF patients without P. aeruginosa infection were characterized by elevated activity of sputum elastase, reduced response of peripheral blood lymphocytes to PHA and significant resistance to the antiproliferative action of glucocorticoids. These parameters were normalized after antibiotic treatment. The patients with prolonged P. aeruginosa infection demonstrated extremely high levels of elastase activity and elevated amounts of sputum IL-8 and TNF-alpha. Although antibiotic treatment resulted in clinical improvement, it failed to suppress excessive immune response in the lung. The data indicate that CF patients with prolonged P. aeruginosa need the modified treatment, which should include immunomodulating drugs and protease inhibitors as well as antibacterial therapy.     

In vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia

Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia , but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na⁺ and K⁺, but not Cl⁻, are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.     

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.     

Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens

The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2) rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.     

Inter- and intraclonal diversity of the Pseudomonas aeruginosa proteome manifests within the secretome

The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation.     

Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.     

Innate immunity mediated by TLR5 as a novel antiinflammatory target for cystic fibrosis lung disease

Novel therapies to target lung inflammation are predicted to improve the lives of people with cystic fibrosis (CF) but specific antiinflammatory targets have not been identified. The goal of this study was to establish whether TLR5 signaling is the key molecular pathway mediating lung inflammation in CF, and to determine whether strategies to inhibit TLR5 can reduce the damaging inflammatory response. The innate immune responses were analyzed in both airway epithelial cells and primary PBMCs from CF patients and matched controls. Additionally, 151 clinical isolates of Pseudomonas aeruginosa from CF patients were assessed for motility and capacity to activate TLR5. Blood and airway cells from CF patients produced significantly more proinflammatory cytokine than did control cells following exposure to the CF pathogens P. aeruginosa and Burkholderia cepacia complex (p < 0.001). Stimulation with pure TLR ligands demonstrated that TLR signaling appears to mediate the excessive cytokine production occurring in CF. Using complementary approaches involving both neutralizing Ab targeting TLR5 and flagellin-deficient bacteria, we established that inhibition of TLR5 abolished the damaging inflammatory response generated by CF airway cells following exposure to P. aeruginosa (p < 0.01). The potential therapeutic value of TLR5 inhibition was further supported by our demonstration that 75% of clinical isolates of P. aeruginosa retained TLR5 activating capacity during chronic CF lung infection. These studies identify the innate immune receptor TLR5 as a novel antiinflammatory target for reducing damaging lung inflammation in CF.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Prevalence of streptococci and increased polymicrobial diversity associated with cystic fibrosis patient stability

Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.     

Neutrophil extracellular trap (NET)-mediated killing of Pseudomonas aeruginosa: evidence of acquired resistance within the CF airway, independent of CFTR

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.     

A bacterial cell to cell signal in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.