The genus Prevotella in cystic fibrosis airways

Airway disease resulting from chronic bacterial colonization and consequential inflammation is the leading cause of morbidity and mortality in patients with Cystic Fibrosis (CF). Although traditionally considered to be due to only a few pathogens, recent re-examination of CF airway microbiology has revealed that polymicrobial communities that include many obligate anaerobes colonize lower airways. The purpose of this study was to examine Prevotella species in CF airways by quantitative culture and phenotypic characterization. Expectorated sputum was transferred to an anaerobic environment immediately following collection and examined by quantitative microbiology using a variety of culture media. Isolates were identified as facultative or obligate anaerobes and the later group was identified by 16S rRNA sequencing. Prevotella spp. represented the majority of isolates. Twelve different species of Prevotella were recovered from 16 patients with three species representing 65% of isolates. Multiple Prevotella species were often isolated from the same sputum sample. These isolates were biochemically characterized using Rapid ID 32A kits (BioMérieux), and for their ability to produce autoinducer-2 and β-lactamases. Considerable phenotypic variability between isolates of the same species was observed. The quantity and composition of Prevotella species within a patients’ airway microbiome varied over time. Our results suggest that the diversity and dynamics of Prevotella in CF airways may contribute to airway disease.     

Surface-lining layer of airways in cystic fibrosis mice

Lung disease is the major cause of death in individuals suffering from cystic fibrosis (CF), with abnormal lung-lining fluids occurring as early as early infancy. However, the precise etiology of CF lung disease is still poorly understood. We investigated the structural components of the airway surface-lining layer in targeted Cftrtm1HGU/Cftrtm1HGU mutant mice and non-CF controls. Five lungs per animal group were fixed by intravascular triple perfusion. The ultrastructure of the surface-lining layer of large and small intrapulmonary conducting airways was systematically investigated according to a standard protocol in transmission and scanning electron micrographs. In both animal groups, the surface-lining layer consisted of an aqueous phase and an osmiophilic film of variable thickness at the air-fluid interface. The aqueous phase usually did extend <1 microm beyond the uppermost tips of the epithelial cells in both animal groups. The aqueous phase of the small airways was slightly more electron dense in Cftrtm1HGU/Cftrtm1HGU than in non-CF mice. Neither the ultrastructure of the surfactant film at the air-fluid interface nor the forms assumed by the osmiophilic structures associated with surfactant turnover in the aqueous layer differed significantly in Cftrtm1HGU/Cftrtm1HGU and non-CF mice. Hence, there were no signs of any ultrastructural abnormalities in the surface-lining layer of young adult Cftrtm1HGU/Cftrtm1HGU mice before infection with CF-related pathogens.     

Primary inflammation in human cystic fibrosis small airways

Most cystic fibrosis (CF) patients die of lung failure, due to the combined effects of bacterial infection, neutrophil-mediated inflammation, and airway obstruction by hyperviscous mucus. To this day, it remains unclear where and how this pathological vicious circle is initiated in vivo. In particular, it has proven difficult to investigate whether inflammatory pathways are dysregulated in CF airways independently of infection. Also, the relative involvement of large (tracheobronchial) vs. small (bronchiolar) airways in CF pathophysiology is still unclear. To help address these issues, we used an in vivo model based on the maturation of human fetal CF and non-CF small airways in severe combined immunodeficiency mice. We show that uninfected mature CF small airway grafts, but not matched non-CF controls, undergo time-dependent neutrophil-mediated inflammation, leading to progressive lung tissue destruction. This model of mature human small airways provides the first clear-cut evidence that, in CF, inflammation may arise at least partly from a primary defect in the regulation of neutrophil recruitment, independently of infection.     

Genetic adaptation of Pseudomonas aeruginosa to the airways of cystic fibrosis patients is catalyzed by hypermutation

In previous work (E. E. Smith, D. G. Buckley, Z. Wu, C. Saenphimmachack, L. R. Hoffman, D. A. D'Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson, Proc. Natl. Acad. Sci. USA 103:8487-8492, 2006) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.     

Characterization of inflammatory reaction in upper airways of cystic fibrosis patients

Inflammatory cell populations have not been yet precisely evaluated in cystic fibrosis (CF) airways. We intended to characterize morphological modifications, inflammatory cell infiltration and cell proliferation in nasal tissues obtained from 15 CF patients and from 6 non-CF patients with nasal polyposis. Morphological analysis showed an intense inflammatory infiltration in CF and non-CF tissues with only few modifications in the epithelium from CF tissues. Inflammatory cell populations characterized by specific immunolabeling were quantified, showing a predominance of macrophages and T- and B-lymphocytes and only moderate numbers of neutrophils in CF tissues; in non-CF polyps, lymphocytes and eosinophils were abundant. Proliferating cell percentages quantified after proliferating cell nuclear antigen immunolabeling were 5.3+/-4.1% (mean +/- SD) in CF polyps and 3.1+/-1.2% in non-CF polyps in epithelium but were very low in lamina propria. Intense inflammation in nasal tissues from CF patients is therefore dominated by macrophages and lymphocytes rather than by neutrophils. While morphology is preserved, proliferation is high in epithelium from CF polyps. These findings should be regarded in the future for a better understanding of inflammation in CF airway disease.     

Dysregulation of TIM-3-galectin-9 pathway in the cystic fibrosis airways

The T-cell Ig and mucin domain-containing molecules (TIMs) have emerged as promising therapeutic targets to correct abnormal immune function in several autoimmune and chronic inflammatory conditions. It has been reported that proinflammatory cytokine dysregulation and neutrophil-dominated inflammation are the main causes of morbidity in cystic fibrosis (CF). However, the role of TIM receptors in CF has not been investigated. In this study, we demonstrated that TIM-3 is constitutively overexpressed in the human CF airway, suggesting a link between CF transmembrane conductance regulator (CFTR) function and TIM-3 expression. Blockade of CFTR function with the CFTR inhibitor-172 induced an upregulation of TIM-3 and its ligand galectin-9 in normal bronchial epithelial cells. We also established that TIM-3 serves as a functional receptor in bronchial epithelial cells, and physiologically relevant concentrations of galectin-9 induced TIM-3 phosphorylation, resulting in increased IL-8 production. In addition, we have demonstrated that both TIM-3 and galectin-9 undergo rapid proteolytic degradation in the CF lung, primarily because of neutrophil elastase and proteinase-3 activity. Our results suggest a novel intrinsic defect that may contribute to the neutrophil-dominated immune response in the CF airways.     

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.     

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.     

Metabolism of P2 receptor agonists in human airways: implications for mucociliary clearance and cystic fibrosis

Extracellular nucleotides are among the most potent mediators of mucociliary clearance (MCC) in human lungs. However, clinical trials revealed that aerosolized nucleotides provide only a transient improvement of MCC to patients diagnosed with cystic fibrosis (CF). In this study, we identified the mechanism that eliminates extracellular nucleotides from human airways. Polarized primary cultures of human bronchial epithelial cells were impermeable to extracellular nucleotides but rapidly dephosphorylated ATP into ADP, AMP, and adenosine. The half-life of a therapeutic ATP concentration (0.1 mm) was approximately 20 s within the periciliary liquid layer. The mucosal epithelial surface eliminated P2 receptor agonists (ATP = UTP > ADP > UDP) at 3-fold higher rates than the serosal surface. We also showed that mucosal (not serosal) ectoATPase activity increases toward areas most susceptible to airway obstruction (nose < bronchi < bronchioles). Bronchial cultures from patients with CF, primary ciliary dyskinesia, or alpha1-antitrypsin deficiency exhibited 3-fold higher mucosal (not serosal) ectoATPase activity than normal cultures. Time course experiments indicated that CF enhances ATP elimination and adenosine accumulation on the mucosal surface. Furthermore, nonspecific alkaline phosphatase was identified as the major regulator of airway nucleotide concentrations in CF, primary ciliary dyskinesia, and alpha1-antitrypsin deficiency. The ectoAT-Pase activity and mRNA expression of mucosally restricted nonspecific alkaline phosphatase were 3-fold higher on bronchial cultures from these patients than from healthy subjects. This study demonstrates that the duration of nucleotide-mediated MCC is limited by epithelial ectonucleotidases throughout human airways, with the efficiency of this mechanism enhanced in chronic inflammatory lung diseases, including CF.     

Dysfunction of the non-neuronal cholinergic system in the airways and blood cells of patients with cystic fibrosis

The non-neuronal cholinergic system is widely expressed in human airways, skin and immune cells. Choline acetyltransferase (ChAT), acetylcholine and nicotine/muscarine receptors are demonstrated in epithelial surface cells, submucosal glands, airway smooth muscle fibres and immune cells. Moreover, acetylcholine is involved in the regulation of cell functions like proliferation, differentiation, migration, organization of the cytoskeleton, cell-cell contact, secretion and transport of ions and water. Cystic fibrosis (CF), the most frequent genetic disorder, is known to be caused by a mutation of the CF-gene coding for the cystic fibrosis transmembrane regulator protein (CFTR). CFTR represents a regulating transport protein for ion channels and processes involving endo- and exocytosis. Despite the identification of the genetic mutation knowledge of the underlying cellular pathways is limited. In the present experiments the cholinergic system was investigated in the peripheral blood and in the lung of CF patients undergoing lung transplantation (n=7). Acetylcholine content in bronchi and lung parenchyma of CF was reduced by 70% compared to controls (tumor-free tissue obtained from patients with lung tumor; n=13). In contrast, ChAT activity was elevated to some extent (p>0.05) in CF, and esterase activity did not differ from control. Acetylcholine content extracted from peripheral leucocytes (30 ml) was also reduced by 70% in CF (n=13) compared to healthy volunteers (n=9). Double labelling experiments with anti-CF antibodies and anti-ChAT antibodies showed a co-localization in peripheral lymphocytes, giving first evidence that CFTR may be linked with the intracellular storage/transport of non-neuronal acetylcholine. It is concluded that the non-neuronal cholinergic system is involved in the pathogenesis of CF. A reduced content of non-neuronal acetylcholine could contribute to the deleterious changes of epithelial ion and water movements in CF, because acetylcholine stimulates apical Cl(-) secretion, inhibits apical Na(+) and water absorption and therewith facilitates mucociliary clearance.     

Microbial ecology and biodiversity in permafrost

Permafrost represents 26% of terrestrial soil ecosystems; yet its biology, essentially microbiology, remains relatively unexplored. The permafrost environment is considered extreme because indigenous microorganisms must survive prolonged exposure to subzero temperatures and background radiation for geological time scales in a habitat with low water activity and extremely low rates of nutrient and metabolite transfer. Yet considerable numbers and biodiversity of bacteria exist in permafrost, some of which may be among the most ancient viable life on Earth. This review describes the permafrost environment as a microbial habitat and reviews recent studies examining microbial biodiversity found in permafrost as well as microbial growth and activity at ambient in situ subzero temperatures. These investigations suggest that functional microbial ecosystems exist within the permafrost environment and may have important implications on global biogeochemical processes as well as the search for past or extant life in permafrost presumably present on Mars and other bodies in our solar system.     

Microbial population genomics and ecology

The origins of biological complexity in microbial ecosystems are encoded within the collective genomes of the community. Cultivation-independent genomic studies provide direct access to the genomes of naturally occurring microbes, cultivated or not. Genome-enabled approaches are now significantly advancing current knowledge of genome content, diversity, population biology and evolution in natural microbial populations.     

Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi

To better understand how airways produce thick airway mucus, nonvolatile solids were measured in liquid secreted by bronchi from normal pig, cystic fibrosis (CF) human, and non-CF human lungs. Bronchi were exposed to various secretagogues and anion secretion inhibitors to induce a range of liquid volume secretion rates. In all three groups, the relationship of solids concentration (percent nonvolatile solids) to liquid volume secretion rate was curvilinear, with higher solids concentration associated with lower rates of liquid volume secretion. In contrast, the secretion rates of solids mass and water mass as functions of liquid volume secretion rates exhibited positive linear correlations. The y-intercepts of the solids mass-liquid volume secretion relationships for all three groups were positive, thus accounting for the higher solids concentrations in airway liquid at low rates of secretion. Predictive models derived from the solids mass and water mass linear equations fit the experimental percent solids data for the three groups. The ratio of solids mass secretion to liquid volume secretion was 5.2 and 2.4 times higher for CF bronchi than for pig and non-CF bronchi, respectively. These results indicate that normal pig, non-CF human, and CF human bronchi produce a high-percent-solids mucus (>8%) at low rates of liquid volume secretion (≤1.0 μl·cm(-2)·h(-1)). However, CF bronchi produce mucus with twice the percent solids (~8%) of pig or non-CF human bronchi at liquid volume secretion rates ≥4.0 μl·cm(-2)·h(-1).     

Proteome analysis reveals adaptation of Pseudomonas aeruginosa to the cystic fibrosis lung environment

Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell, membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pattern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to PAO revealed differential expression of virulence factors under non-inducing conditions. The membrane proteome of strain C showed modulation of the expression of porins involved in nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed retention of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether, our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat during the course of chronic colonization.