Major Cystic Fibrosis Research Articles

The bacterial serine protease inhibitor ecotin inhibits neutrophil elastase enzymatic activity in cystic fibrosis sputa

Cystic Fibrosis (CF) airway disease is characterized by impaired mucociliary clearance, chronic, polymicrobial infections and robust, neutrophil-dominated inflammation. Pulmonary disease is the leading cause of morbidity and mortality in people with CF and is due to progressive airflow obstruction and ultimately respiratory failure. One of the earliest abnormalities in CF airway disease is the recruitment of neutrophils to the lungs. Neutrophil activation leads to the release of their intracellular content, including neutrophil elastase (NE), that damages lung tissues in CF. Our goal is to characterize a known bacterial NE inhibitor, ecotin, in the CF airway environment. Our results indicate that ecotins cloned from four Gram-negative bacterial species (Campylobacter rectus, Campylobacter showae, Escherichia coli and Pseudomonas aeruginosa) inhibit NE activity in CF sputum samples in a dose-dependent manner. Although we observed differences in the NE-inhibitory activity of the tested ecotins with the Campylobacter homologs being the most effective in NE inhibition in CF sputa, none of the ecotins impaired the ability of human neutrophils to kill major CF respiratory pathogens, P. aeruginosa or S. aureus, in vitro. Overall, we demonstrate that bacterial ecotins inhibit NE activity in CF sputa without compromising bacterial killing by neutrophils.

An oral commensal attenuates Pseudomonas aeruginosa-induced airway inflammation and modulates nitrite flux in respiratory epithelium.

Polymicrobial airway infections in persons with cystic fibrosis (pwCF) can positively or negatively impact the course of disease. A major CF pathogen, Pseudomonas aeruginosa, often establishes a chronic infection leading to lung deterioration. Interestingly, the presence of certain oral commensal streptococci is correlated with improved outcomes for pwCF. We previously reported that hydrogen peroxide production by these commensals combined with nitrite generates reactive nitrogen intermediates (RNI), which inhibit P. aeruginosa in vitro. In this study, we utilized a rat co-infection lung model to assess whether oral commensal-generated RNI can restrict the pathogenesis of a CF isolate of P. aeruginosa. We report that the oral commensal Streptococcus parasanguinis and nitrite reduce P. aeruginosa-induced host inflammation in wild-type rats. To better recapitulate CF-specific airway conditions, we used a bronchial epithelial cell culture model to gain a better understanding of how S. parasanguinis and nitrite may influence P. aeruginosa burden during a CF infection. Hence, we co-infected wild-type and cystic fibrosis transconductance regulator (CFTR) channel-deficient bronchial epithelial cells with P. aeruginosa and S. parasanguinis with or without nitrite. Strikingly, S. parasanguinis reduced the bacterial burden of P. aeruginosa without nitrite, promoted epithelial cell viability, and stimulated nitrite production in the wild-type and CFTR-deficient epithelial cells, where nitrite induction was more apparent in the CFTR mutant cells. Taken together, our study demonstrates that the commensal S. parasanguinis may provide protection against P. aeruginosa-induced inflammation and cell death, as well as modulate nitrite flux in airway epithelial cells. IMPORTANCE Respiratory infections are a leading cause of morbidity and mortality in people with cystic fibrosis (CF). These infections are polymicrobial in nature with overt pathogens and other colonizing microbes present. Microbiome data have indicated that the presence of oral commensal bacteria in the lungs is correlated with improved outcomes. We hypothesize that one oral commensal, Streptococcus parasanguinis, inhibits CF pathogens and modulates the host immune response. One major CF pathogen is Pseudomonas aeruginosa, a Gram-negative, opportunistic bacterium with intrinsic drug resistance and an arsenal of virulence factors. We have previously shown that S. parasanguinis inhibits P. aeruginosa in vitro in a nitrite-dependent manner through the production of reactive nitrogen intermediates. In this study, we demonstrate that while this mechanism is evident in a cell culture model of the CF airway, an alternative mechanism by which S. parasanguinis may improve outcomes for people with CF is through immunomodulation.

Variable Susceptibility to Gallium Compounds of Major Cystic Fibrosis Pathogens.

The decreasing efficacy of existing antibiotics against pulmonary pathogens that affect cystic fibrosis (CF) patients calls for the development of novel antimicrobials. Iron uptake and metabolism are vital processes for bacteria, hence potential therapeutic targets. Gallium [Ga(III)] is a ferric iron-mimetic that inhibits bacterial growth by disrupting iron uptake and metabolism. In this work we evaluate the efficacy of three Ga(III) compounds, namely, Ga(NO3)3, (GaN), Ga(III)-maltolate (GaM), and Ga(III)-protoporphyrin IX (GaPPIX), against a collection of CF pathogens using both reference media and media mimicking biological fluids. All CF pathogens, except Streptococcus pneumoniae, were susceptible to at least one Ga(III) compound. Notably, Mycobacterium abscessus and Stenotrophomonas maltophilia were susceptible to all Ga(III) compounds. Achromobacter xylosoxidans, Burkholderia cepacia complex, and Pseudomonas aeruginosa were more susceptible to GaN and GaM, whereas Staphylococcus aureus and Haemophilus influenzae were more sensitive to GaPPIX. The results of this study support the development of Ga(III)-based therapy as a broad-spectrum strategy to treat CF lung infections.

Outcomes of artery embolisation for cystic fibrosis patients with haemoptysis: a 20‐year experience at a major Australian tertiary centre

There are no published data on Australian adult cystic fibrosis (CF) patient outcomes post bronchial arterial embolisation (BAE). We report 20 years of experience of BAE at a major Australian tertiary adult CF centre, where 46 patients underwent 100 BAE during this period. Mortality rate was comparable to previous studies (4% per year) and most who died had repeat BAE requirements. A higher proportion (9 out of 45) of patients were transplanted compared to previous publications. Repeat BAE was common and significantly higher in patients already on tranexamic acid.

Discrete choice experiment (DCE) to quantify the influence of trial features on the decision to participate in cystic fibrosis (CF) clinical trials

IntroductionEngaging people with cystic fibrosis (CF) in clinical trials is critical to improving outcomes for this fatal disease. Following extensive exploration of engagement in CF trials we believe six key...

Burkholderia ubonensis High-Level Tetracycline Resistance Is Due to Efflux Pump Synergy Involving a Novel TetA(64) Resistance Determinant.

Burkholderia ubonensis, a nonpathogenic soil bacterium belonging to the Burkholderia cepacia complex (Bcc), is highly resistant to some clinically significant antibiotics. The concern is that B. ubonensis may serve as a resistance reservoir for Bcc or B. pseudomallei complex (Bpc) organisms that are opportunistic human pathogens. Using a B. ubonensis strain highly resistant to tetracycline (MIC, ≥256 µg/ml), we identified and characterized tetA(64) that encodes a novel tetracycline-specific efflux pump of the major facilitator superfamily. TetA(64) and associated TetR(64) regulator expression are induced by tetracyclines. Although TetA(64) is the primary tetracycline and doxycycline resistance determinant, maximum tetracycline and doxycycline resistance requires synergy between TetA(64) and the nonspecific AmrAB-OprA resistance nodulation cell division efflux pump. TetA(64) does not efflux minocycline, tigecycline, and eravacycline. Comprehensive screening of genome sequences showed that TetA(64) is unequally distributed in the Bcc and absent from the Bpc. It is present in some major cystic fibrosis pathogens, like Burkholderia cenocepacia, but absent from others like Burkholderia multivorans The tetR(64)-tetA(64) genes are located in a region of chromosome 1 that is highly conserved in Burkholderia sp. Because there is no evidence for transposition, the tetR(64)-tetA(64) genes may have been acquired by homologous recombination after horizontal gene transfer. Although Burkholderia species contain a resident multicomponent efflux pump that allows them to respond to tetracyclines up to a certain concentration, the acquisition of the single-component TetA(64) by some species likely provides the synergy that these bacteria need to defend against high tetracycline concentrations in niche environments.

Antibiotic susceptibility of cystic fibrosis lung microbiome members in a multispecies biofilm

The lungs of cystic fibrosis (CF) patients are often chronically colonized by multiple microbial species that can form biofilms, including the major CF pathogen Pseudomonas aeruginosa. Herewith, lower microbial diversity in CF airways is typically associated with worse health outcomes. In an attempt to treat CF lung infections patients are frequently exposed to antibiotics, which may affect microbial diversity. This study aimed at understanding if common antibiotics that target P. aeruginosa influence microbial diversity. To this end, a microaerophilic multispecies biofilm model of frequently co-isolated members of the CF lung microbiome (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus anginosus, Achromobacter xylosoxidans, Rothia mucilaginosa, and Gemella haemolysans) was exposed to antipseudomonal antibiotics. We found that antibiotics that affected several dominant species (i.e. ceftazidime, tobramycin) resulted in higher species evenness compared to colistin, which is only active against P. aeruginosa. Furthermore, susceptibility of individual species in the multispecies biofilm following antibiotic treatment was compared to that of the respective single-species biofilms, showing no differences. Adding three anaerobic species (Prevotella melaninogenica, Veillonella parvula, and Fusobacterium nucleatum) to the multispecies biofilm did not influence antibiotic susceptibility. In conclusion, our study demonstrates antibiotic-dependent effects on microbial community diversity of multispecies biofilms comprised of CF microbiome members.

Antimicrobial Treatment Provides a Competitive Advantage to Mycobacterium abscessus in a Dual-Species Biofilm with Pseudomonas aeruginosa.

The physiological factors that contribute to Mycobacterium abscessus lung infections remain unclear. We determined whether antibiotic treatment targeting a major cystic fibrosis pathogen (i.e., Pseudomonas aeruginosa) could provide the ideal conditions for the establishment of M. abscessus infection. Our data showed that P. aeruginosa inhibited M. abscessus biofilm formation under control conditions and that antimicrobial therapy selectively targeting P. aeruginosa diminished this competitive interaction, thereby increasing M. abscessus survival.

SLC9A3 Affects Vas Deferens Development and Associates with Taiwanese Congenital Bilateral Absence of the Vas Deferens.

Background The pathophysiology of Taiwanese congenital bilateral absence of the vas deferens (CBAVD) is different from that in Caucasians. In particular, major cystic fibrosis transmembrane conductance regulator (CFTR) mutations and cystic fibrosis are absent in the former. Instead, deficiency in solute carrier family 9 sodium/hydrogen exchanger isoform 3 (SLC9A3) may play a role by generating obstructive azoospermia and degraded epithelial structure in the reproductive tract. Objectives The objective of the study was to test whether SLC9A3 variants cause Taiwanese CBAVD. Materials and Methods Six-month-old Slc9a3−/−male mice were used to evaluate the effect of long-term SLC9A3 loss on the reproductive system. A case-control cohort of 29 men with CBAVD and 32 fertile men were genotyped for SLC9A3 variants. Results SLC9A3 was expressed and localized in the apical border of the epithelium of human vas deferens and glandular epithelium of the seminal vesicle. SLC9A3 deficiency specifically induces atrophy of vas deferens and unfolding of seminal vesicle mucosa in mice. Loss of SLC9A3 increased the incidence of CBAVD in humans from 3.1% to 37.9% (p < 0.001). Up to 75.9% of CBAVD patients carry at least one variant in either SLC9A3 or CFTR. Discussion Our findings build upon previous data associated with CBAVD pathogenesis. Here, we now report for the first time an association between CBAVD and loss of SLC9A3 and propose that specific defects in the reproductive duct due to SLC9A3 variants drive CBAVD development. Conclusion The data implicate loss of SLC9A3 as a basis of Taiwanese CBAVD and highlight SLC9A3 function in reproduction.

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.

SLC6A14, an amino acid transporter, modifies the primary CF defect in fluid secretion.

The severity of intestinal disease associated with Cystic Fibrosis (CF) is variable in the patient population and this variability is partially conferred by the influence of modifier genes. Genome-wide association studies have identified SLC6A14, an electrogenic amino acid transporter, as a genetic modifier of CF-associated meconium ileus. The purpose of the current work was to determine the biological role of Slc6a14, by disrupting its expression in CF mice bearing the major mutation, F508del. We found that disruption of Slc6a14 worsened the intestinal fluid secretion defect, characteristic of these mice. In vitro studies of mouse intestinal organoids revealed that exacerbation of the primary defect was associated with reduced arginine uptake across the apical membrane, with aberrant nitric oxide and cyclic GMP-mediated regulation of the major CF-causing mutant protein. Together, these studies highlight the role of this apical transporter in modifying cellular nitric oxide levels, residual function of the major CF mutant and potentially, its promise as a therapeutic target.

Influence of three-dimensional lung epithelial cells and interspecies interactions on antibiotic efficacy against Mycobacterium abscessus and Pseudomonas aeruginosa.

Mycobacterium abscessus lung infection is a major health problem for cystic fibrosis (CF) patients. Understanding the in vivo factors that influence the outcome of therapy may help addressing the poor correlation between in vitro and in vivo antibiotic efficacy. We evaluated the influence of interspecies interactions and lung epithelial cells on antibiotic efficacy. Therefore, single and dual-species biofilms of M. abscessus and a major CF pathogen (Pseudomonas aeruginosa) were cultured on a plastic surface or on in vivo-like three-dimensional (3-D) lung epithelial cells, and the activity of antibiotics (colistin, amikacin, clarithromycin, ceftazidime) in inhibiting biofilm formation was evaluated. Using the most physiologically relevant model (dual-species biofilms on 3-D cells), we observed that treatment with antibiotics during biofilm development inhibited P. aeruginosa but not M. abscessus biofilms, resulting in a competitive advantage for the latter. Clarithromycin efficacy against P. aeruginosa was inhibited by 3-D lung cells. In addition, biofilm induction of M. abscessus was observed by certain antibiotics on plastic but not on 3-D cells. Pseudomonas aeruginosa influenced the efficacy of certain antibiotics against M. abscessus, but not vice versa. In conclusion, these results suggest a role of host cells and interspecies interactions in bacterial responses to antimicrobials.

Correctors of the Major Cystic Fibrosis Mutant Interact through Membrane-Spanning Domains.

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.

Changing Rates of Chronic Pseudomonas aeruginosa Infections in Cystic Fibrosis: A Population-Based Cohort Study.

Chronic Pseudomonas aeruginosa lung infection is associated with significant morbidity and mortality in cystic fibrosis (CF). It is not known whether recent advances in care have affected the rates of chronic infection. We aimed to determine if the rates of developing new chronic P. aeruginosa infection among adolescents and adults with CF significantly changed over time. The cohort consisted of individuals with CF followed in the Cystic Fibrosis Foundation Patient Registry aged ≥13 years without chronic P. aeruginosa at baseline. Multivariable regression models accounting for within-patient correlation were used to assess the change in rate of developing chronic P. aeruginosa infection between 2003 and 2012. A total of 15504 individuals were followed for a median of 5 (interquartile range, 2-9) years. The annual rates of developing new chronic P. aeruginosa decreased from 14.3% in 2003 to 6.4% in 2012. After adjusting for potential confounders, relative risk (RR) of developing chronic P. aeruginosa infection decreased significantly over time compared to 2003 (P value test of trend < .001). Compared with 2003, the RR of developing chronic P. aeruginosa infection in 2012 was 0.33 (95% confidence interval, 0.30-0.37). No significant increases in risk of chronic infections with other major CF bacterial pathogens relative to 2003 were identified. Among individuals with CF, a significant decrease in the risk and rates of developing chronic P. aeruginosa infection between 2003 and 2012 was observed. Whether this decline results in changes in clinical outcomes warrants further exploration.

The secreted polyketide boydone A is responsible for the anti-Staphylococcus aureus activity of Scedosporium boydii.

Usually living as a soil saprophyte, the filamentous fungus Scedosporium boydii may also cause various infections in human. Particularly, it is one of the major causative agents of fungal colonization of the airways in patients with cystic fibrosis (CF). To compete with other microorganisms in the environment, fungi have evolved sophisticated strategies, including the production of secondary metabolites with antimicrobial activity that may also help them to establish successfully within the respiratory tract of receptive hosts. Here, the culture filtrate from a human pathogenic strain of S. boydii was investigated searching for an antibacterial activity, mainly against the major CF bacterial pathogens. A high antibacterial activity against Staphylococcus aureus, including methicillin-resistant strains of this species, was observed. Bio-guided fractionation and analysis of the active fractions by nuclear magnetic resonance or by high-performance liquid chromatography and high-resolution electrospray ionization-mass spectrometry allowed us to identify boydone A as responsible for this antibacterial activity. Together, these results suggest that this six-membered cyclic polyketide could be one of the virulence factors of the fungus. Genes involved in the synthesis of this secreted metabolite are currently being identified in order to confirm the role of this polyketide in pathogenesis.

Orkambi® and amplifier co‐therapy improves function from a rare CFTR mutation in gene‐edited cells and patient tissue

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508. It has been predicted that Orkambi® could treat patients with rarer mutations of similar “theratype”; however, a standardized approach confirming efficacy in these cohorts has not been reported. Here, we demonstrate that patients bearing the rare mutation: c.3700 A>G, causing protein misprocessing and altered channel function—similar to ΔF508‐CFTR, are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor, respectively, this combination led to a minor in vitro response in patient‐derived tissue. A CRISPR/Cas9‐edited bronchial epithelial cell line bearing this mutation enabled studies showing that an “amplifier” compound, effective in increasing the levels of immature CFTR protein, augmented the Orkambi® response. Importantly, this “amplifier” effect was recapitulated in patient‐derived nasal cultures—providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF‐causing mutation. We propose that this multi‐disciplinary approach, including creation of CRISPR/Cas9‐edited cells to profile modulators together with validation using primary tissue, will facilitate therapy development for patients with rare CF mutations.

Adherence of Pseudomonas aeruginosa to cystic fibrosis buccal epithelial cells.

Pseudomonas aeruginosa cystic fibrosis buccal adherence assay http://ow.ly/lPtB306VKIQ

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2–4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD–exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases.

Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants.

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

The Role of Bacterial Secretion Systems in the Virulence of Gram-Negative Airway Pathogens Associated with Cystic Fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disorder in Caucasians. It is caused by mutation of the CF transmembrane conductance regulator (CFTR) gene. A defect in the CFTR ion channel causes a dramatic change in the composition of the airway surface fluid, leading to a highly viscous mucus layer. In healthy individuals, the majority of bacteria trapped in the mucus layer are removed and destroyed by mucociliary clearance. However, in the lungs of patients with CF, the mucociliary clearance is impaired due to dehydration of the airway surface fluid. As a consequence, patients with CF are highly susceptible to chronic or intermittent pulmonary infections, often causing extensive lung inflammation and damage, accompanied by a decreased life expectancy. This mini review will focus on the different secretion mechanisms used by the major bacterial CF pathogens to release virulence factors, their role in resistance and discusses the potential for therapeutically targeting secretion systems.