Consequences of cystic fibrosis transmembrane regulator mutations on inflammatory cells

Recent studies in cystic fibrosis (CF) transmembrane regulator (CFTR) mutations and function have shed light on its involvement in disease progression. The extent of cell and tissue distribution of CFTR facilitates systemic dysfunction of ion transport in patients carrying a mutation in CFTR, however, its incidences as cofounding risk factor to develop other diseases is not well studied. In this review we differentiate the dysfunctions driven by CFTR mutations in cell of the immune system and their role in CF progression and examine the types of medical treatments available to patients up to date.Fil: Grumelli, Sandra. Universidad Católica de Córdoba; ArgentinaFil: Islan, German Abel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Castro, Guillermo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Bacterial Host Interactions in Cystic Fibrosis

Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as the host response persists, progressive destruction of the normal architecture of lung tissue ensues with catastrophic consequences for the host

Consequences of cystic fibrosis transmembrane regulator mutations on inflammatory cells

Recent studies in cystic fibrosis (CF) transmembrane regulator (CFTR) mutations and function have shed light on its involvement in disease progression. The extent of cell and tissue distribution of CFTR facilitates systemic dysfunction of ion transport in patients carrying a mutation in CFTR, however, its incidences as cofounding risk factor to develop other diseases is not well studied. In this review we differentiate the dysfunctions driven by CFTR mutations in cell of the immune system and their role in CF progression and examine the types of medical treatments available to patients up to date.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Examining the Role of Specific Virulence Mechanisms During Pseudomonas Aeruginosa Infection in a Zebrafish Model of Cystic Fibrosis

Cystic fibrosis (CF) is the most common lethal hereditary disease. CF is caused by recessive mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is associated with multi-­-organ defects resulting from improper ion transport across epithelial membranes. Chronic lung infection by the environmentally ubiquitous opportunistic human pathogen Pseudomonas aeruginosa and the subsequent hyperinflammation that occurs as the host immune system combats the bacterium cause substantial morbidity and mortality in CF. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were also found to display a iv significant iron deficiency (ID) compared to control embryos, a symptom commonly diagnosed in CF patients. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, a phenomenon that was not observed with any of the other bacterial species examined. The bacterial burden in Cftr morphants infected with a P. aeruginosa LasR mutant, a quorum sensing (QS)-­-deficient strain, was comparable to that in control fish indicating that the regulation of virulence factors through QS is required for enhancement of infection in the absence of Cftr. Cftr morphants were then challenged with P. aeruginosa mutants defective in the expression of QS regulated virulence factors. A mutant defective in the production of Exotoxin A (ETA) resulted in similar bacterial clearance in both the Cftr morphant and control embryos. The reduction in directed neutrophil migration to a P. aeruginosa infection was also restored when zebrafish embryos were challenged with the ETA mutant. Taken together, these data point towards a possible explanation for the specificity between P. aeruginosa and CFTR. The zebrafish system provides a multitude of advantages for studying the unique pathophysiology resulting from defective expression of CFTR, investigating the pathogenesis of P. aeruginosa and elucidating the role that the innate system plays in the host response to acute bacterial infections commonly associated with cystic fibrosis

Challenges and opportunities in the development of novel antimicrobial therapeutics for cystic fibrosis

Chronic respiratory infection is the primary driver of mortality in individuals with cystic fibrosis (CF). Existing drug screening models utilised in preclinical antimicrobial development are unable to mimic the complex CF respiratory environment. Consequently, antimicrobials showing promising activity in preclinical models often fail to translate through to clinical efficacy in people with CF. Model systems used in CF anti-infective drug discovery and development range from antimicrobial susceptibility testing in nutrient broth, through to 2D and 3D in vitro tissue culture systems and in vivo models. No single model fully recapitulates every key aspect of the CF lung. To improve the outcomes of people with CF (PwCF) it is necessary to develop a set of preclinical models that collectively recapitulate the CF respiratory environment to a high degree of accuracy. Models must be validated for their ability to mimic aspects of the CF lung and associated lung infection, through evaluation of biomarkers that can also be assessed following treatment in the clinic. This will give preclinical models greater predictive power for identification of antimicrobials with clinical efficacy. The landscape of CF is changing, with the advent of modulator therapies that correct the function of the CFTR protein, while antivirulence drugs and phage therapy are emerging alternative treatments to chronic infection. This review discusses the challenges faced in current antimicrobial development pipelines, including the advantages and disadvantages of current preclinical models and the impact of emerging treatments

Animal models of cystic fibrosis

AbstractAnimal models of cystic fibrosis, in particular several different mutant mouse strains obtained by homologous recombination, have contributed considerably to our understanding of CF pathology. In this review, we describe and compare the main phenotypic features of these models. Recent and possible future developments in this field are discussed

Novel agents with inhibitory activity against the Burkholderia cepacia complex

Pulmonary bacterial infections account for 95% of morbidity and mortality in cystic fibrosis (CF) patients, and include a limited spectrum of bacteria; Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, and members of the Burkholderia cepacia complex (Bcc). "B. cepacia" was first recognised in the late 1970's as a cause of life threatening respiratory infections in CF. Initial clinical observations noted that 20% of colonised CF patients developed "cepacia syndrome", a rapid and fatal necrotising pneumonia. In addition, epidemiological evidence highlighted the potential spread of certain "B. cepacia''' strains, that most isolates were highly resistant to conventional antibiotics and consequently, Bcc infections are untreatable. Subsequent taxonomic studies have identified the Bcc that contains ten distinct species ofbacteria previously termed "5. cepacia". Clinical distribution ofBcc species in CF is restricted to mainly B. cenocepacia (50%), B. multivorans (38%) and B. vietnamiensis (7%)The aim of this study was to investigate novel antimicrobial agents against the Bcc and other problematic and emerging CF associated bacterial pathogens including multi-resistant epidemic P. aeruginosa strains, methicillin resistant S. aureus (MRSA), and Stenotrophomonas maltophilia. The novel antimicrobial strategies examined were based on three main themes: First, the use of natural honey, second, the potential use of bacteriophage and their associated lytic enzymes and third, novel mammalian cationic ß-defensins. The project utilised a vast collection of bacterial isolates and included relevant clinical, environmental and epidemic strains. The susceptibility to conventional antibiotics was measured, and resistance was shown to vary across the Bcc. In general, clinical isolates were statistically more resistant to conventional antibiotics than environmental isolates.Members of the Bcc, and an extended panel ofresistant organisms were shown to be sensitive to New Zealand manuka honey (NZMh). The MICs ranged from 9 to 17% (w/v), and the MBCs ranged from 9 to 20% (w/v). The antimicrobial component of NZMh was investigated, and focussed on osmolality, pH and H₂O₂. All were found to contribute to the antibacterial activity, although none were solely responsible for the activity. Killing-curves suggested that NZMh kills within 24 hours. The NZMh preparation was applied to a CF patient infected with B. cenocepacia J2315, and clinical data highlighted possible benefits to the patient.Novel Bcc specific bacteriophages were identified from environmental samples and from lysogeny studies. The spectrum of activity of the novel bacteriophages, and previously reported Bcc bacteriophage (NS1 and NS2), was determined using a panel comprising 66 isolates of the Bcc, 55 isolates representing other pseudomonads, and 40 B. pseudomallei strains. The novel phages were shown to be very promiscuous and had activity across the Bcc, with some active against P. aeruginosa, B. gladioli, and B. pseudomallei. The wide spectrum of activity was detrimental to therapeutic use, therefore, the phage-encoded lytic enzymes were the focus for further study: bacteriophage therapy with a novel twist. Two enzymes were investigated: the B. cepacia bacteriophage Bcep781 endolysin and the P. aeruginosa phage D3 endolysin. The Bcep781 phage and phage DNA was not available, therefore the endolysin gene was synthesized using recursive PCR. Briefly, twenty-two overlapping oligonucleotides encoding the entire gene were synthesized and constructed into the endolysin gene using a single PCR reaction. Both genes were cloned into an expression plasmid and the enzymes were recombinantly expressed as 6-His fusion proteins in BL21 E. coli cells. Bcep781 endolysin was purified using nickel-affinity chromatography, and the D3 lysin was purified using a Resource S® purification protocol. High-resolution mass spectrometry analysis highlighted discrepancies in both lysins, and neither proved to active against relevant bacteria tested.The activity of cationic antimicrobial peptides (CAMPs) including: a synthetic novel murine ß-defensin (Defrl) with 5-cysteine residues, which forms a covalently bound dimer; its 6-cysteine analog (Def-cys); a chemically reduced Defrl; polymyxin B and colistin; were assessed against the Bcc, as well as several multi-resistant bacterial CF pathogens. Two Bcc isolates, B. cepacia type strain ATCC 25416 and B. cenocepacia type strain J2315, were found to be inherently resistant to all CAMPs utilised in this study. Epidemic P. aeruginosa isolates were found to have a MIC of 6 μg/ml for Defrl and a MIC of 50-100 μg/ml for Def-cys, suggesting a possible relationship between defensin structure and function. Similarly, the MIC of 6 μg/ml was also noted for S. maltophilia and Ralstonia sp. that were found to be resistant to polymyxin B and colistin. The recombinant production of Defrl was also attempted in a number of expression systems in E. coli. However, although Defrl was successfully expressed, the recombinant proteins were highly insoluble. This study showed that resistance varies within the Bee. However, the data show that NZMh exerts a bactericidal effect on members of the Bcc, including B. cenocepacia J2315, and that such activity may be utilised clinically. The novel Bcc bacteriophage may prove to be a useful panel for further study, either as vectors for horizontal gene transfer or as therapeutic agents. The data confirm previous observations that the Bcc are inherently resistant to CAMPs, including a novel 5- cysteine defensin. Despite this finding, synthetic Defrl was shown to be active against a panel of multi-resistant pathogens associated with infections in CF. Further research is required to optimise the recombinant expression of Bcep781 endolysin, D3 lysin, and Defrl, to enable their use in the treatment of multiply resistant infections in CF and the wider hospital environment