Effect of Aspergillus fumigatus and Candida albicans on pro-inflammatory response in cystic fibrosis epithelium

AbstractBackgroundThe identification of filamentous fungi and/or yeasts in the airway secretions of individuals with cystic fibrosis (CF) is becoming increasingly prevalent; yet the importance of these organisms in relation to underlying inflammation is poorly defined.MethodsCystic fibrosis bronchial epithelial cells (CFBE) and human bronchial epithelial cells (HBE) were co-incubated with Candida albicans whole cells or Aspergillus fumigatus conidia for 24h prior to the measurement of pro-inflammatory cytokines IL-6 and IL-8 by ELISA.ResultsTreatment of HBE or CFBE with C. albicans whole cells did not alter cytokine secretion. However treatment of CFBE with A. fumigatus conidia resulted in a 1.45-fold increase in IL-6 and a 1.65-fold increase in IL-8 secretion in comparison to basal levels; in contrast there was far less secretion from HBE cells.ConclusionOur data indicate that A. fumigatus infection modulates a pro-inflammatory response in CF epithelial cells while C. albicans does not

A Selective Irreversible Inhibitor of Furin Does Not Prevent Pseudomonas Aeruginosa Exotoxin A-Induced Airway Epithelial Cytotoxicity

Many bacterial and viral pathogens (or their toxins), including Pseudomonas aeruginosa exotoxin A, require processing by host pro-protein convertases such as furin to cause disease. We report the development of a novel irreversible inhibitor of furin (QUB-F1) consisting of a diphenyl phosphonate electrophilic warhead coupled with a substrate-like peptide (RVKR), that also includes a biotin tag, to facilitate activity-based profiling/visualisation. QUB-F1 displays greater selectivity for furin, in comparison to a widely used exemplar compound (furin I) which has a chloromethylketone warhead coupled to RVKR, when tested against the serine trypsin-like proteases (trypsin, prostasin and matriptase), factor Xa and the cysteine protease cathepsin B. We demonstrate QUB-F1 does not prevent P. aeruginosa exotoxin A-induced airway epithelial cell toxicity; in contrast to furin I, despite inhibiting cell surface furin-like activity to a similar degree. This finding indicates additional proteases, which are sensitive to the more broad-spectrum furin I compound, may be involved in this process

Bacterial proteases and haemostasis dysregulation in the CF lung.

BACKGROUND: Pathogenic bacteria which chronically colonise the cystic fibrosis (CF) lung produce a number of virulence determinants, including distinct proteolytic activities. The potential role bacterial proteases play on haemostatic dysregulation within the CF lung is, however, poorly defined, despite haemoptysis being a common complication in CF. METHODS: The potential impact of known CF pathogens (Pseudomonas aeruginosa and Burkholderia cepacia complex spp.) on haemostasis was examined for their ability to degrade fibrinogen and dysregulate fibrin clot formation and platelet aggregation. RESULTS: Results demonstrate that key CF pathogens growing as a biofilm on mucin exhibit considerable fibrinogenolytic activity, resulting in fibrinogen breakdown, impaired clot formation, and modulation of platelet aggregation. Human neutrophil elastase may also contribute to fibrinogen breakdown and dysregulated clot formation at high concentration. CONCLUSION: Bacterial-derived proteases may play an important role in the dysregulation of airway haemostasis, and potentially contribute to episodes of haemoptysis within the CF lung.Funding was provided by a grant to LM, AMcD and JSE from the CF Trust, UK (Big Lottery Funding/UK CF Microbiology Consortium) which supported GE (PhD studentship). MM was funded by a Department of Education and Learning (DEL), Northern Ireland PhD studentship

Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

The key metabolic regulator, AMP-activated protein kinase (AMPK) is reported to be downregulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491 respectively as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues

A single sulfatase is required to access colonic mucin by a gut bacterium

International audienceHumans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel diseas

Airborne indoor allergen serine proteases and their contribution to sensitisation and activation of innate immunity in allergic airway disease

Common airborne allergens (pollen, animal dander and those from fungi and insects) are the main triggers of type I allergic disorder in the respiratory system and are associated with allergic rhinitis, allergic asthma, as well as immunoglobulin E (IgE)-mediated allergic bronchopulmonary aspergillosis. These allergens promote IgE crosslinking, vasodilation, infiltration of inflammatory cells, mucosal barrier dysfunction, extracellular matrix deposition and smooth muscle spasm, which collectively cause remodelling of the airways. Fungus and insect (house dust mite and cockroaches) indoor allergens are particularly rich in proteases. Indeed, more than 40 different types of aeroallergen proteases, which have both IgE-neutralising and tissue-destructive activities, have been documented in the Allergen Nomenclature database. Of all the inhaled protease allergens, 85% are classed as serine protease activities and include trypsin-like, chymotrypsin-like and collagenolytic serine proteases. In this article, we review and compare the allergenicity and proteolytic effect of allergen serine proteases as listed in the Allergen Nomenclature and MEROPS databases and highlight their contribution to allergic sensitisation, disruption of the epithelial barrier and activation of innate immunity in allergic airways disease. The utility of small-molecule inhibitors of allergen serine proteases as a potential treatment strategy for allergic airways disease will also be discussed.<br/

Novel Inhibitors and Activity-Based Probes Targeting Trypsin-Like Serine Proteases

The trypsin-like proteases (TLPs) play widespread and diverse roles, in a host of physiological and pathological processes including clot dissolution, extracellular matrix remodelling, infection, angiogenesis, wound healing and tumour invasion/metastasis. Moreover, these enzymes are involved in the disruption of normal lung function in a range of respiratory diseases including allergic asthma where several allergenic proteases have been identified. Here, we report the synthesis of a series of peptide derivatives containing an N-alkyl glycine analogue of arginine, bearing differing electrophilic leaving groups (carbamate and triazole urea), and demonstrate their function as potent, irreversible inhibitors of trypsin and TLPs, to include activities from cockroach extract. As such, these inhibitors are suitable for use as activity probes (APs) in activity-based profiling (ABP) applications

Novel inhibitors and activity-based probes targeting serine proteases

Serine proteases play varied and manifold roles in important biological, physiological, and pathological processes. These include viral, bacterial, and parasitic infection, allergic sensitization, tumor invasion, and metastasis. The use of activity-based profiling has been foundational in pinpointing the precise roles of serine proteases across this myriad of processes. A broad range of serine protease-targeted activity-based probe (ABP) chemotypes have been developed and we have recently introduced biotinylated and “clickable” peptides containing P1 N-alkyl glycine arginine N-hydroxy succinimidyl (NHS) carbamates as ABPs for detection/profiling of trypsin-like serine proteases. This present study provides synthetic details for the preparation of additional examples of this ABP chemotype, which function as potent irreversible inhibitors of their respective target serine protease. We describe their use for the activity-based profiling of a broad range of serine proteases including trypsin, the trypsin-like protease plasmin, chymotrypsin, cathepsin G, and neutrophil elastase (NE), including the profiling of the latter protease in clinical samples obtained from patients with cystic fibrosis

Furin as a therapeutic target in cystic fibrosis airways disease

Clinical management of cystic fibrosis (CF) has been greatly improved by the development of small molecule modulators of the CF transmembrane conductance regulator (CFTR). These drugs help to address some of the basic genetic defects of CFTR; however, no suitable CFTR modulators exist for 10% of people with CF (PWCF). An alternative, mutation-agnostic therapeutic approach is therefore still required. In CF airways, elevated levels of the proprotein convertase furin contribute to the dysregulation of key processes that drive disease pathogenesis. Furin plays a critical role in the proteolytic activation of the epithelial sodium channel; hyperactivity of which causes airways dehydration and loss of effective mucociliary clearance. Furin is also responsible for the processing of transforming growth factor-β, which is increased in bronchoalveolar lavage fluid from PWCF and is associated with neutrophilic inflammation and reduced pulmonary function. Pathogenic substrates of furin include Pseudomonas exotoxin A, a major toxic product associated with Pseudomonas aeruginosa infection and the spike glycoprotein of severe acute respiratory syndrome coronavirus 2, the causative pathogen for coronavirus disease 2019. In this review we discuss the importance of furin substrates in the progression of CF airways disease and highlight selective furin inhibition as a therapeutic strategy to provide clinical benefit to all PWCF