Multi-omics study of keystone species in the cystic fibrosis lung microbiome

Abstract Background: Ecological networking and in vitro studies have predicted that anaerobic, mucus-degrading bacteria are keystone species in cystic fibrosis (CF) microbiomes by sustaining the growth of canonical CF pathogens. Here, a multi-omics approach was deployed to test this hypothesis in vivo and in real time during a transition in antibiotic therapy of a CF patient with a hypervariable lung function phenotype . Results: Quantitative meta-omics and community culturing demonstrated that the use of a non-traditional clindamycin therapy targeting gram-positives and gram-negative anaerobes re-structured the entire CF microbial community. During rapid lung function loss, when the patient was off antibiotics, the microbial community was dominated by anaerobic mucus-degrading Streptococcus sp., Veilonella sp., and Prevotella sp. that produced fermentation gas and led to the accumulation of fermentation products in sputum. The rise of anaerobes was followed within 6 days by an increase in Pseudomonas aeruginosa transcripts encoding the acquisition of fermentation products from anaerobes and the production of virulence factors. The initiation of clindamycin treatment reduced the fermentation and the abundance of anaerobes. Clindamycin also lowered the abundance and transcription of P. aeruginosa, which is resistant to this antibiotic. The treatment stabilized the patient’s lung function and improved respiratory health for two months, lengthening by a factor of four the between-hospitalization time for this patient. Conclusions: The results presented here show that killing anaerobes, the weakest link in the community in terms of antibiotic resistance, effectively limited the growth of classic CF pathogen by disrupting community cross-feeding. The role that anaerobic, mucus-degrading bacteria played in structuring the CF microbiome corroborates in vivo their position as keystone bacteria, with high impact on community function despite lower relative abundances.

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Multi-Omics Study of Keystone Species in a Cystic Fibrosis Microbiome

International Journal of Molecular Sciences ◽

10.3390/ijms222112050 ◽

2021 ◽

Vol 22 (21) ◽

pp. 12050

Author(s):

Cynthia B. Silveira ◽

Ana G. Cobián-Güemes ◽

Carla Uranga ◽

Jonathon L. Baker ◽

Anna Edlund ◽

...

Keyword(s):

Cystic Fibrosis ◽

Lung Function ◽

Sequence Data ◽

Anaerobic Bacteria ◽

Keystone Species ◽

Clinical Diagnostics ◽

Epifluorescence Microscopy ◽

Fermentation Products ◽

Degrading Bacteria

Ecological networking and in vitro studies predict that anaerobic, mucus-degrading bacteria are keystone species in cystic fibrosis (CF) microbiomes. The metabolic byproducts from these bacteria facilitate the colonization and growth of CF pathogens like Pseudomonas aeruginosa. Here, a multi-omics study informed the control of putative anaerobic keystone species during a transition in antibiotic therapy of a CF patient. A quantitative metagenomics approach combining sequence data with epifluorescence microscopy showed that during periods of rapid lung function loss, the patient’s lung microbiome was dominated by the anaerobic, mucus-degrading bacteria belonging to Streptococcus, Veillonella, and Prevotella genera. Untargeted metabolomics and community cultures identified high rates of fermentation in these sputa, with the accumulation of lactic acid, citric acid, and acetic acid. P. aeruginosa utilized these fermentation products for growth, as indicated by quantitative transcriptomics data. Transcription levels of P. aeruginosa genes for the utilization of fermentation products were proportional to the abundance of anaerobic bacteria. Clindamycin therapy targeting Gram-positive anaerobes rapidly suppressed anaerobic bacteria and the accumulation of fermentation products. Clindamycin also lowered the abundance and transcription of P. aeruginosa, even though this patient’s strain was resistant to this antibiotic. The treatment stabilized the patient’s lung function and improved respiratory health for two months, lengthening by a factor of four the between-hospitalization time for this patient. Killing anaerobes indirectly limited the growth of P. aeruginosa by disrupting the cross-feeding of fermentation products. This case study supports the hypothesis that facultative anaerobes operated as keystone species in this CF microbiome. Personalized multi-omics may become a viable approach for routine clinical diagnostics in the future, providing critical information to inform treatment decisions.

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Modified hCFTRmRNA restores normal lung function in a mouse model of cystic fibrosis

10.1101/202853 ◽

2017 ◽

Cited By ~ 1

Author(s):

AKM Ashiqul Haque ◽

Alexander Dewerth ◽

Justin S. Antony ◽

Joachim Riethmüller ◽

Ngadhnjim Latifi ◽

...

Keyword(s):

Cystic Fibrosis ◽

Lung Function ◽

Messenger Rna ◽

Viral Vectors ◽

Therapeutic Option ◽

Normal Lung ◽

Monogenic Disease ◽

Promising Therapeutic Approach

AbstractBeing a classic monogenic disease, gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary effective drug. We thus explored the expression of human (h)CFTR encoded by hCFTRcmRNAin vitro, analyzed by flow cytometry and Western Blot and its function with a YFP assay. Very similar effects could be observedin vivowhen hCFTRcmRNA was assembled with Chitosan-coated PLGA to nanoparticles (NPs) and intratracheally (i.t.) or intravenously (i.v) injected, the latter one as an alternative administration route to circumvent the clogged airways of CF patients. This significantly improved lung function, which suggests that hCFTRcmRNA-NPs are a promising therapeutic option for CF patients independent of theirCFTRgenotype.

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Faculty Opinions recommendation of Identification of new small molecule inhibitors of cystic fibrosis transmembrane conductance regulator protein: in vitro and in vivo studies.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1069767.522696 ◽

2007 ◽

Author(s):

Kim Barrett

Keyword(s):

Cystic Fibrosis ◽

Small Molecule ◽

Small Molecule Inhibitors ◽

In Vivo Studies ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Regulator Protein ◽

Cystic Fibrosis Transmembrane

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Preliminary Report of In vitro and In vivo Effectiveness of Dornase Alfa on SARS-CoV-2 Infection (Preprint)

10.2196/preprints.20200 ◽

2020 ◽

Author(s):

Hacer Kuzu Okur ◽

Koray Yalcin ◽

Cihan Tastan ◽

Sevda Demir ◽

Bulut Yurtsever ◽

...

Keyword(s):

Cystic Fibrosis ◽

Respiratory Tract ◽

Mononuclear Cells ◽

Multiple Organ ◽

Peripheral Blood Mononuclear ◽

Dornase Alfa ◽

Tract Infections ◽

Adult Peripheral Blood

UNSTRUCTURED Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). Coronavirus disease 2019 (COVID-19) pandemic affected more than two million people over the world, resulting in unprecedented health, social and economic crises. The COVID-19, viral pneumonia that progresses to ARDS and even multiple organ failure, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and predict severe respiratory diseases. A similar mucus structure is detected in COVID-19 patients due to the accumulation of excessive NET in the lungs. Here, we show our preliminary results with dornase alfa that may have an in-vitro anti-viral effect against SARS-CoV-2 infection in a bovine kidney cell line, MDBK without drug toxicity on healthy adult peripheral blood mononuclear cells. In this preliminary study, we also showed that dornase alfa can promote clearance of NET formation in both an in-vitro and three COVID-19 cases who showed clinical improvement in radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea and coughing.

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Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

International Journal of Molecular Sciences ◽

10.3390/ijms22052530 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2530

Author(s):

Bijean D. Ford ◽

Diego Moncada Giraldo ◽

Camilla Margaroli ◽

Vincent D. Giacalone ◽

Milton R. Brown ◽

...

Keyword(s):

Cystic Fibrosis ◽

Bactericidal Activity ◽

Scavenger Receptor ◽

Receptor Expression ◽

Blood Monocytes ◽

Bacterial Killing ◽

Blood Neutrophils ◽

Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

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Prevalence and impact of oprD mutations in Pseudomonas aeruginosa strains in cystic fibrosis

10.1101/19013870 ◽

2019 ◽

Author(s):

Laura J. Sherrard ◽

Bryan A. Wee ◽

Christine Duplancic ◽

Kay A. Ramsay ◽

Keyur A. Dave ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Function ◽

Hazard Rate ◽

Carbapenem Resistance ◽

Adverse Outcomes ◽

Nonsense Mutations ◽

Allelic Variants ◽

Novel Variants

ABSTRACTDefective OprD porins contribute to carbapenem resistance and may be important in Pseudomonas aeruginosa adaptation to cystic fibrosis airways. It is unclear whether oprD mutations are fixed in populations of shared strains that are transmitted between patients or whether novel variants arise during infection. We investigated oprD sequences and antimicrobial resistance of two common Australian shared strains, constructed P. aeruginosa mutants with the most common oprD allelic variants and compared characteristics between patients with or without evidence of infection with strains harbouring these variants. Our data show that three independently acquired nonsense mutations arising from a 1-base pair substitution are fixed in strain sub-lineages. These nonsense mutations are likely to contribute to reduced carbapenem susceptibility in the sub-lineages without compromising in vitro fitness. Not only was lung function worse among patients infected with strains harbouring the nonsense mutations than those without, but they also had an increased hazard rate of lung transplantation/death. Our findings further highlight that understanding adaptive changes may help to distinguish patients with greater adverse outcomes despite infection with the same strain.

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Type-4 Phosphodiesterase (PDE4) Blockade Reduces NETosis in Cystic Fibrosis

Frontiers in Pharmacology ◽

10.3389/fphar.2021.702677 ◽

2021 ◽

Vol 12 ◽

Author(s):

Licia Totani ◽

Concetta Amore ◽

Antonio Piccoli ◽

Giuseppe Dell’Elba ◽

Angelo Di Santo ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Lung Disease ◽

Pde4 Inhibitor ◽

Free Dna ◽

Impaired Lung Function ◽

Cellular Integrity ◽

Net Release

Neutrophilic inflammation is a key determinant of cystic fibrosis (CF) lung disease. Neutrophil-derived free DNA, released in the form of extracellular traps (NETs), significantly correlates with impaired lung function in patients with CF, underlying their pathogenetic role in CF lung disease. Thus, specific approaches to control NETosis of neutrophils migrated into the lungs may be clinically relevant in CF. We investigated the efficacy of phosphodiesterase (PDE) type-4 inhibitors, in vitro, on NET release by neutrophils from healthy volunteers and individuals with CF, and in vivo, on NET accumulation and lung inflammation in mice infected with Pseudomonas aeruginosa. PDE4 blockade curbed endotoxin-induced NET production and preserved cellular integrity and apoptosis in neutrophils, from healthy subjects and patients with CF, challenged with endotoxin, in vitro. The pharmacological effects of PDE4 inhibitors were significantly more evident on CF neutrophils. In a mouse model of Pseudomonas aeruginosa chronic infection, aerosol treatment with roflumilast, a selective PDE4 inhibitor, gave a significant reduction in free DNA in the BALF. This was accompanied by reduced citrullination of histone H3 in neutrophils migrated into the airways. Roflumilast-treated mice showed a significant improvement in weight recovery. Our study provides the first evidence that PDE4 blockade controls NETosis in vitro and in vivo, in CF-relevant models. Since selective PDE4 inhibitors have been recently approved for the treatment of COPD and psoriasis, our present results encourage clinical trials to test the efficacy of this class of drugs in CF.

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Food Starch Structure Impacts Gut Microbiome Composition

mSphere ◽

10.1128/msphere.00086-18 ◽

2018 ◽

Vol 3 (3) ◽

Cited By ~ 44

Author(s):

Frederick J. Warren ◽

Naoki M. Fukuma ◽

Deirdre Mikkelsen ◽

Bernadine M. Flanagan ◽

Barbara A. Williams ◽

...

Keyword(s):

Small Intestine ◽

Microbial Community ◽

Gut Microbiome ◽

Nutritive Value ◽

Human Diet ◽

Fermentation Products ◽

Microbiome Composition ◽

Starch Fermentation ◽

Starch Structure

ABSTRACT Starch is a major source of energy in the human diet and is consumed in diverse forms. Resistant starch (RS) escapes small intestinal digestion and is fermented in the colon by the resident microbiota, with beneficial impacts on colonic function and host health, but the impacts of the micro- and nanoscale structure of different physical forms of food starch on the broader microbial community have not been described previously. Here, we use a porcine in vitro fermentation model to establish that starch structure dramatically impacts microbiome composition, including the key amylolytic species, and markedly alters both digestion kinetics and fermentation outcomes. We show that three characteristic food forms of starch that survive digestion in the small intestine each give rise to substantial and distinct changes in the microbiome and in fermentation products. Our results highlight the complexity of starch fermentation processes and indicate that not all forms of RS in foods are degraded or fermented in the same way. This work points the way for the design of RS with tailored degradation by defined microbial communities, informed by an understanding of how substrate structure influences the gut microbiome, to improve nutritive value and/or health benefits. IMPORTANCE Dietary starch is a major component in the human diet. A proportion of the starch in our diet escapes digestion in the small intestine and is fermented in the colon. In this study, we use a model of the colon, seeded with porcine feces, in which we investigate the fermentation of a variety of starches with structures typical of those found in foods. We show that the microbial community changes over time in our model colon are highly dependent on the structure of the substrate and how accessible the starch is to colonic microbes. These findings have important implications for how we classify starches reaching the colon and for the design of foods with improved nutritional properties.

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Murine colonic mucosa hyperproliferation. I. Elevated CFTR expression and enhanced cAMP-dependent Cl−secretion

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2000.278.5.g753 ◽

2000 ◽

Vol 278 (5) ◽

pp. G753-G764 ◽

Cited By ~ 23

Author(s):

Shahid Umar ◽

Jason Scott ◽

Joseph H. Sellin ◽

William P. Dubinsky ◽

Andrew P. Morris

Keyword(s):

Cystic Fibrosis ◽

Fluid Transport ◽

Cellular Proliferation ◽

Neck Region ◽

Anion Channel ◽

Cellular Level ◽

Cystic Fibrosis Gene ◽

Mrna And Protein Expression

Fluid transport in the large intestine is mediated by the cystic fibrosis gene product and cAMP-dependent anion channel cystic fibrosis transmembrane conductance regulator (CFTR). cAMP-mediated Cl−secretion by gastrointestinal cell lines in vitro has been positively correlated with the insertion of CFTR into the apical membrane of differentiated senescent colonocytes and negatively correlated with the failure of CFTR to insert into the plasma membrane of their undifferentiated proliferating counterparts. In native tissues, this relationship remains unresolved. We demonstrate, in a transmissible murine colonic hyperplasia (TMCH) model, that (8-fold) colonocyte proliferation was accompanied by increased cellular CFTR mRNA and protein expression (8.3- and 2.4-fold, respectively) and enhanced mucosal cAMP-dependent Cl−secretion (2.3-fold). By immunofluorescence microscopy, cellular CFTR expression was restricted to the apical pole of cells at the base of the epithelial crypt. In contrast, increased cellular proliferation in vivo led to increases in both the cellular level and the total number of cells expressing this anion channel, with cellular CFTR staining extending into the crypt neck region. Hyperproliferating colonocytes accumulated large amounts of CFTR in apically oriented subcellular perinuclear compartments. This novel mode of CFTR regulation may explain why high endogenous levels of cellular CFTR mRNA and protein within the TMCH epithelium were not matched with larger increases in transmucosal CFTR Cl−current.

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