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

A shotgun metaproteomics approach to study the microbiome of cystic fibrosis (CF) patients

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Faecal (meta)proteomics : a tool to investigate dysbiosis and inflammation in patients with cystic fibrosis

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Development of a new hazelnut sandwich ELISA based on detection of Cor a 9, a major hazelnut allergen

The emerging health problem of food-induced allergic reactions, including life-threatening anaphylaxis, presents an important challenge to the food. In the framework of the current ALLERRISK project, analytical approaches for allergen detection are evaluated and developed. Cor a 9 is a major hazelnut protein with nutrient reservoir function, and that also known as a major food allergen. Presence of Cor a 9 indicates contamination of a product with hazelnut and, consequently, potential risk of allergenicity. Crude extract of hazelnut was obtained from milled mixture of hazelnuts from 10 commercially available brands. Then the extract was concentrated and applied to gel filtration and Con A affinity chromatography columns for Cor a 9 isolation and purification. The final product was characterized by analytical gel permeation chromatography, SDS PAGE and MALDI mass spectrometry. The purified Cor a 9 was used as an antigen for the production of polyclonal antibodies in chickens according to the protocol developed in our laboratory. A part of the most active antibodies was further purified by gel permeation chromatography and characterized by SDS PAGE before further use. The prepared antibodies were applied for development of a sandwich ELISA using polyclonal antibody-enzyme adducts as a tracer. Optimization of reagent concentrations led us to achieve detection limit ~10 ng/ml. Influence of ionic strength, pH and buffer composition on analytical parameters of the developed ELISA were thoroughly studied. The developed ELISA can be successfully applied for quantitative detection of Cor a 9 in large pH (pH = 5 – 10) and ionic strength (0.05 – 1.5 M salts solution) ranges. Cross-reactivity with series of nuts, ovalbumin, whey, and wheat proteins was also investigated (Figure 2) and showed that the developed assay was very specific. Moreover, presence of several other proteins (peanut, cashew, ovalbumin, whey and wheat) at high concentrations (1 mg/ml) in the presence of Cor a 9 did not influence the calibration curve of Cor a 9. Detection of hazelnut protein in cookies with known amount of hazelnut inside initial dough showed good recovery (35-40%) and absence of significant signal in blank samples (i.e. without spiking of hazelnut)

The bacterial antitoxin HipB establishes a ternary complex with operator DNA and phosphorylated toxin HipA to regulate bacterial persistence

Nearly all bacteria exhibit a type of phenotypic growth described as persistence that is thought to underlie antibiotic tolerance and recalcitrant chronic infections. The chromosomally encoded high-persistence (Hip) toxin-antitoxin proteins HipA(SO) and HipB(SO) from Shewanella oneidensis, a proteobacterium with unusual respiratory capacities, constitute a type II toxin-antitoxin protein module. Here we show that phosphorylated HipA(SO) can engage in an unexpected ternary complex with HipB(SO) and double-stranded operator DNA that is distinct from the prototypical counterpart complex from Escherichia coli. The structure of HipB(SO) in complex with operator DNA reveals a flexible C-terminus that is sequestered by HipA(SO) in the ternary complex, indicative of its role in binding HipA(SO) to abolish its function in persistence. The structure of HipA(SO) in complex with a non-hydrolyzable ATP analogue shows that HipA(SO) autophosphorylation is coupled to an unusual conformational change of its phosphorylation loop. However, HipA(SO) is unable to phosphorylate the translation factor Elongation factor Tu, contrary to previous reports, but in agreement with more recent findings. Our studies suggest that the phosphorylation state of HipA is an important factor in persistence and that the structural and mechanistic diversity of HipAB modules as regulatory factors in bacterial persistence is broader than previously thought

Whey proteins interact with lipids during autoxidation primarly via the production of reactive carbonyl species

Nowadays, it is well recognized that polyunsaturated fatty acids (PUFAs) can provide extensive nutritional and health benefits. Thus, ω-3 PUFAs have been considered to contribute to the prevention of several diseases. Recognition of the potential benefits of ω-3 fatty acids has stimulated increased interest towards the fortification of foods with oils rich in these particular fatty acids. However, enrichment of food products with such unsaturated fatty acids should be carefully evaluated since they are highly susceptible to oxidation. Moreover, exposure of proteins to peroxidizing lipids or their secondary breakdown products may induce severe changes in proteins, including polymerization, insolubilization and formation of lipid – protein complexes. Several amino acids, but mainly mainly cysteine, methionine, histidine, tyrosine and lysine are affected by the secondary lipid oxidation products, therefore leading to reductions of their availability. The objective of this study was to characterize changes induced in whey protein isolate (a mixture of α-lactalbumin and β-lactoglobulin) through oxidation with oils with different unsaturation degrees and different initial oxidation status. The incubation of whey proteins with oils caused an increase in protein bound carbonyl content through the interaction of lipid oxidation products with the amino acids. Changes in the amino acid composition were therefore also observed mostly upon incubation with fish and highly oxidized soybean oil. Interaction of proteins with lipid oxidation products was taking place via interaction of the reactive carbonyl species formed thus leading to protein aggregates formation. Protein aggregation was therefore one of the most prominent consequence of the interaction of whey proteins with oxidizing lipids

Microfluidics-based liquid chromatography/mass spectrometry multiple reaction monitoring approach for the relative quantification of Burkholderia cenocepacia secreted virulence factors

Rationale: Burkholderia cenocepacia is an opportunistic pathogen that is commonly isolated from patients with cystic fibrosis (CF). Quorum sensing has been suggested to play a role in the activity of type II and type VI secretion systems and the release of virulence factors. Apart from the classical acyl homoserine lactone quorum sensing, B. cenocepacia also uses the diffusible signal factor system (DSF). Quantitative information on the true impact of DSF molecules on the release of ZmpA and other virulence factors is lacking. Methods: Based on results of a label-free proteomics analysis addressing changes in the secretome in response to DSFs, a panel of peptides was selected to develop a microfluidics liquid chromatography/mass spectrometry (LC/MS) method implementing single reaction monitoring (SRM) to quantify B. cenocepacia virulence factors. Results: Increase in secretion of virulence factors upon treatment with BDSF was observed for ZmpA and Aida, but not for ZmpB. Type VI secretion system dependent Hcp1 and TecA were decreased. However, non-physiological amounts of BDSF were needed to provoke the effect. DSFs from P. aeruginosa and S. maltophilia were also affecting virulence factor secretion, but the effect was smaller than for the endogenous BDSF. Conclusions: Microfluidics-based SRM is a useful tool to quantitatively assess the impact of quorum sensing on the release of virulence factors by (opportunistic) pathogens

Stability of whey protein derived peptides upon severe protein glycation

Cow’s milk and dairy products are major nutrients in the human diet, especially during infancy. Though at one time whey proteins were considered as by-product of the cheese making process, nowadays, due to their wide ranging nutritional, biological, and functional properties, whey proteins are often used in food technology as low-cost protein ingredients. However, whey-protein fractions, such as β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) represent the major allergens in cow’s milk. Therefore, the use of whey proteins in food might pose a serious threat to the milk allergic consumers. It is well reported that food processing may modify the allergenicity and detectability of proteins. This can be due to hydrolysis or chemical reactions with other food components (carbohydrates, fatty acids etc), leading to modification or destruction of the allergen’s structure. Therefore, the objective of this study was in the first stage to investigate the influence of glycation on the molecular changes induced in whey proteins. This was done with a special focus on the modifications induced on the lysine residues, free amino groups, the formation of protein bound carbonyls, formation of fluorescent compounds and brown polymers and on the protein aggregation. Matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) was used to get a better insight into the molecular changes that took place on the protein level. Unexpectedly, this study led to the identification of protein segments in the epitope region that remained unmodified during the experiments that mimic typical food processing conditions. The 57Val – Lys76 and 31Val – Arg56 from β–LG, remained unchanged disregarding the severe heating treatment in the presence of glucose and bulk proteins and they could be identified by either direct MALDI-TOF MS and MS/MS or after a more tedious separation using reversed phase chromatography. It is proposed that these peptide segments can be used as analytical targets for the development of more robust methods for the assessment of the presence of whey proteins in processed foodstuffs. Moreover, MALDI-TOF MS and MS/MS holds potential to be used as a screening tool for the identification of such stable peptides