High nuclease activity of long persisting Staphylococcus aureus isolates within the airways of cystic fibrosis patients protects against NET- mediated killing
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PROVIDER: PRJNA586836 | ENA |
REPOSITORIES: ENA
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Project description:Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.
2019-11-01 | GSE139662 | GEO
Project description: Not available
| S-EPMC6849659 | biostudies-literature
Project description:Staphylococcus aureus is one of the earliest pathogens that persists the airways of cystic fibrosis (CF) patients and contributes to increased inflammation and decreased lung function. In contrast to other staphylococci, S. aureus possesses two superoxide dismutases (SODs), SodA and SodM, with SodM being unique to S. aureus. Both SODs arm S. aureus for its fight against oxidative stress, a byproduct of inflammatory reactions. Despite complex investigations it is still unclear, if both enzymes are crucial for the special pathogenicity of S. aureus. To investigate the role of both SODs during staphylococcal persistence in CF airways, we analyzed survival and gene expression of S. aureus CF isolates and laboratory strains in different CF-related in vitro and ex vivo settings. Bacteria located in inflammatory and oxidized CF sputum transcribed high levels of sodA and sodM. Especially expression values of sodM were remarkably higher in CF sputum than in bacterial in vitro cultures. Interestingly, also S. aureus located in airway epithelial cells expressed elevated transcript numbers of both SODs, indicating that S. aureus is exposed to oxidative stress at various sites within CF airways. Both enzymes promoted survival of S. aureus during PMN killing and seem to act compensatory, thereby giving evidence that the interwoven interaction of SodA and SodM contributes to S. aureus virulence and facilitates S. aureus persistence within CF airways
2020-01-27 | GSE131862 | GEO
Project description:Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain. We asked what abnormality impairs elimination when a bacterium lands on the pristine surface of a newborn CF airway? To investigate this defect, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO3- transport. Without CFTR, airway epithelial HCO3- secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF and that assaying ASL pH or bacterial killing could report on the benefit of therapeutic interventions. 11 samples of trachea primary airway epithelial cultures representing CFTR+/+ and CFTR-/- pigs. Pig samples representing 14 bronchus and 12 trachea tissue samples submitted in GSE21071.
2012-03-29 | E-GEOD-36906 | biostudies-arrayexpress
Project description:The goal of this study was to determine the effects of commonly used nuclease treatments and their divalent metal cation cofactors on Pol I occupancy during NET-seq. Other groups have previously used micrococcal nuclease (MNase) + CaCl2 and deoxyribonuclease I (DNase I) + MnCl2 in NET-seq protocols probing for Pol II occupancy. However, the effect of these treatments on polymerase occupancy has not been fully explored. Therefore, we tested the impact of these treatments (CaCl2, CaCl2 + MNase, MnCl2, and MnCl2 + DNase I) on Pol I occupancy in vivo. We found that the exposure of Pol I to either CaCl2 or MnCl2 caused a significant reduction in occupancy on the rDNA template. Nuclease treatment (with either MNase or DNase I) did not cause an additional significant effect on Pol I occupancy in vivo beyond that of either CaCl2 or MnCl2. Finally, we found that MnCl2 treatment caused a more significant reduction in Pol I occupancy as compared to CaCl2, suggesting that perhaps MnCl2 stimulates the intrinsic cleavage activity of Pol I more robustly vs. CaCl2 in vivo. Altogether, these findings indicate that the inclusion of these treatments (especially that of divalent metal cations) in NET-seq protocols for Pol I cause a significant change in the polymerase occupancy.
2022-10-27 | GSE216460 | GEO
Project description:Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method enabling improved identification of PA proteome with CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. We detected 1528 PA proteins (encoded by 1458 core genes and 70 accessory genes) that were expressed in CF airways, of which 1178 proteins were commonly identified with the ex vivo-grown PA populations. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.
2019-05-09 | PXD011792 | Pride
Project description:Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting inflammation cause most of the morbidity and mortality, how loss of CFTR first disrupts airway host defense has remained uncertain. We asked what abnormality impairs elimination when a bacterium lands on the pristine surface of a newborn CF airway? To investigate this defect, we interrogated the viability of individual bacteria immobilized on solid grids and placed on the airway surface. As a model we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly killed bacteria in vivo, when removed from the lung, and in primary epithelial cultures. Lack of CFTR reduced bacterial killing. We found that ASL pH was more acidic in CF, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defense defect to loss of CFTR, an anion channel that facilitates HCO3- transport. Without CFTR, airway epithelial HCO3- secretion is defective, ASL pH falls and inhibits antimicrobial function, and thereby impairs killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF and that assaying ASL pH or bacterial killing could report on the benefit of therapeutic interventions.
2012-03-30 | GSE36906 | GEO
Project description:The 3D model of the connective airways tissue was obtained by using a specific bottom up approach of tissue engineering allowing to develop 3D tissues featured by the presence of fibroblasts embedded into an endogenous matrix produced by fibroblasts themselves.
2020-06-15 | GSE141536 | GEO
Project description:Epigenome analysis of NET samples
| PRJNA553758 | ENA
Project description:Expression profile of human GEP-NET tumors, including 113 fresh frozen biopsies of primary and metastatic tumours originating from pancreas (P-NET, 83 primary and 30 metastasis), 81 from small intestine (SI-NET, 44 primary and 37 metastasis), and 18 from rectum (RE-NET, 3 primary and 15 metastasis).
2018-02-25 | GSE98894 | GEO