Ara h 2-specific IgE epitope-like peptides inhibit the binding of IgE to Ara h 2 and suppress lgE-dependent effector cell activation.

BACKGROUND: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope-paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE-mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2-specific IgE epitope-like peptides that can bind to allergen-specific IgE paratopes and suppress effector cell activation. METHODS: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty-six children or young adults who had peanut allergy were studied. RESULTS: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut-allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p < .001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2-induced effector cell activation (p < .05) and increased the half-maximal Ara h 2 effective concentration (p < .05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. CONCLUSIONS: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE-dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope-paratop blocking.

Silver Fir (Abies alba L.) Polyphenolic Extract Shows Beneficial Influence on Chondrogenesis In Vitro under Normal and Inflammatory Conditions.

Several plant polyphenols have been shown to reduce osteoarthritis symptoms due to their antioxidant, anti-inflammatory and immunomodulatory properties. We investigated the effects of two different polyphenolic extracts (Belinal, Pycnogenol) and two different polyphenols (resveratrol, quercetin) on the chondrogenic potential of bone-derived mesenchymal stem/stromal cells (MSCs) from healthy donors and patients with osteoarthritis. Our main aim was to determine whether Belinal, a commercially available polyphenolic extract from silver fir (Abies alba L.) branches, has comparable chondrogenic potential with the other tested extract and the polyphenols under inflammatory and non-inflammatory conditions. In our study, Belinal promoted significantly greater chondrogenesis compared to the untreated (p = 0.0289) and resveratrol-treated (p = 0.0468) MSCs from patients with hip osteoarthritis under non-inflammatory conditions. Under inflammatory conditions, chondrogenesis was significantly enhanced for MSCs treated with Belinal compared to the control (p = 0.0483). The other extract and the polyphenols did not show any significant effects on chondrogenesis under non-inflammatory or inflammatory conditions. None of the tested extracts and polyphenols showed significant effects on chondrogenesis in healthy donors, under either non-inflammatory or inflammatory conditions. Our data show that Belinal can boost the chondrogenesis of MSCs derived from patients with osteoarthritis, under both non-inflammatory and inflammatory conditions.

Engineered and wild-type L. lactis promote anti-inflammatory cytokine signalling in inflammatory bowel disease patient's mucosa.

Dysbiosis of intestinal microbiota and aberrant inflammatory responses in gastrointestinal mucosa plays important roles in the development of inflammatory bowel disease (IBD). The purpose of this study was to demonstrate the probiotic activity of Lactococcus lactis and the ability of TNF-α-binding by recombinant L. lactis bearing TNF-α-binding affibodies. Various concentrations of recombinant L. lactis were exposed to TNF-α and its binding measured by ELISA. Mucosal biopsies of patients with active IBD were incubated with various L. lactis strains or E. coli DH5α strain and concentrations of TNF-α, IL-23, and IL-10 in the supernatants determined by ELISA. Recombinant L. lactis, at 1 × 109 and 1 × 108 CFU/mL, bound 22.6% and 18.4%, respectively of TNF-α (p < 0.05). When IBD-mucosa was incubated with any L. lactis strain at 1 × 109 CFU/mL, levels of TNF-α and IL-23 were significantly decreased and that of IL-10 increased relative to that for the sterile culture. Opposite trends were observed with E. coli cultures. Recombinant L. lactis at 1 × 108 CFU/mL bound as much as 62.8% (p = 0.026) of TNF-α in IBD-mucosa supernatants compared with the control strain. L. lactis strains are reported, for the first time, to induce an ex vivo anti-inflammatory cytokine profile in IBD inflamed mucosa. L. lactis could therefore constitute a promising alternative approach for treating IBD.

Silver Fir (Abies alba) Extracts Inhibit Enzymes Involved in Blood Glucose Management and Protect against Oxidative Stress in High Glucose Environment.

The diet rich in fruits and vegetables reduces the risk of metabolic syndrome, including diabetes development by various mechanisms of action, mainly due to the presence of polyphenolic compounds. Extracts from different conifer species are known to be a rich source of various polyphenols. In the present study we elucidated the in vitro mechanism of anti-diabetic activity of silver fir (Abies alba) wood and bark extracts and compared their activity to non-coniferous sweet chestnut wood extract and standardized maritime pine bark extract. Extracts and lignans were tested for their inhibitory activity of enzymes involved in the regulation of blood glucose in vitro. The ability of extracts to protect against oxidative stress in high glucose environment was tested on mouse myoblast cell line. Silver fir wood and bark extracts were shown to be effective inhibitors of α-glucosidase, α-amylase and dipeptidyl peptidase 4, three enzymes involved in the regulation of blood glucose levels. Coniferous extracts also showed protection against oxidative stress generated in high glucose environment. Lignans, particularly pinoresinol diglucoside, isolariciresinol and secolariciresinol were shown to be important contributors of antihyperglycemic activity through inhibition of dipeptidyl peptidase 4. This corroborates previously published in vivo results on blood glucose level obtained with silver fir wood extract and supports the use of silver fir wood and bark extracts as food supplements or functional foods in borderline diabetes.

Engineering recombinant Lactococcus lactis as a delivery vehicle for BPC-157 peptide with antioxidant activities.

Lactic acid bacteria (LAB) are attractive hosts for the expression of heterologous proteins and can be engineered to deliver therapeutic proteins or peptides to mucosal surfaces. The gastric stable pentadecapeptide BPC-157 is able to prevent and treat gastrointestinal inflammation by reducing the production of reactive oxygen species (ROS). In this study, we used LAB Lactococcus lactis as a vector to deliver BPC-157 by surface display and trypsin shedding or by secretion to the growth medium. Surface display of BPC-157 was achieved by fusing it with basic membrane protein A (BmpA) or with the peptidoglycan binding domain of AcmA and Usp45 secretion signal. While the expression of BmpA-fusion proteins was higher than that of AcmA/Usp45-fusion protein, the surface display ability of BPC-157 was approximately 14-fold higher with AcmA/Usp45-fusion protein. Release of BPC-157 from the bacterial surface or from isolated fusion proteins by trypsinization was demonstrated with anti-BPC-157 antibodies or by mass spectrometry. The concentration of BPC-157 delivered by surface display via AcmA/Usp45-fusion was 30 ng/ml. This increased to 117 ng/ml by Usp45 signal-mediated secretion, making the latter the most effective lactococcal delivery approach for BPC-157. Secreted BPC-157 significantly decreased ROS production in 149BR fibroblast cell model, suggesting its potential benefit in the treatment of intestinal inflammations. Additionally, a comparison of different modes of small peptide delivery by L. lactis, performed in the present study, will facilitate the future use of L. lactis as peptide delivery vehicle.

Screening of Phenolic Compounds Reveals Inhibitory Activity of Nordihydroguaiaretic Acid Against Three Enzymes Involved in the Regulation of Blood Glucose Level.

In this work we have focused on the inhibition of three different enzymes with a role in postprandial glucose management: α-amylase, α-glucosidase and dipeptidyl peptidase 4. The assortment of 29 monomeric phenolic compounds was first screened at a single concentration. Next, the IC50 values of tested compounds were evaluated for compounds that considerably inhibited any of the enzymes. Nordihydroguaiaretic acid, a phenolic compound abundant in Creosote bush Larrea tridentata, possessed inhibitory activity for all tested enzymes. This in vitro mechanism of action supports traditional use of Creosote bush in diabetes treatment.

Improvement of LysM-mediated surface display of designed ankyrin repeat proteins (DARPins) in recombinant and nonrecombinant strains of Lactococcus lactis and Lactobacillus Species.

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities.

In vivo imaging of Lactococcus lactis, Lactobacillus plantarum and Escherichia coli expressing infrared fluorescent protein in mice.

BACKGROUND: In vivo imaging of orally administered lactic acid bacteria (LAB) and commensal bacteria in mice is shown to provide information on the spatial and temporal distribution of bacteria in the gastrointestinal tract. The bacteria can be detected and monitored using bioluminescence or near-infrared fluorescence. RESULTS: Fluorescence imaging of bacteria was established by expressing the infrared fluorescent protein IRFP713 in Lactococcus lactis, Lactobacillus plantarum and Escherichia coli. All three bacterial species were monitored in live mice and no major differences in transit time were observed. Bacteria passed through the stomach and small intestine in 1 h and the majority were secreted from the large intestine after 6-8 h. Intestinal localization of bacteria was confirmed by imaging the isolated intestines and culturing the intestinal content. The use of fluorescence tomography for spatial localization of fluorescent bacteria has been established. The expression of an additional infrared fluorescent protein IRFP682 enabled concomitant detection of two bacterial populations in live mice. CONCLUSIONS: The present work provides a methodological basis for future studies of probiotic and theranostic actions of LAB in mouse disease models.

Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders.

New treatment approaches for Clostridioides difficile infections: alternatives to antibiotics and fecal microbiota transplantation.

Clostridioides difficile causes a range of debilitating intestinal symptoms that may be fatal. It is particularly problematic as a hospital-acquired infection, causing significant costs to the health care system. Antibiotics, such as vancomycin and fidaxomicin, are still the drugs of choice for C. difficile infections, but their effectiveness is limited, and microbial interventions are emerging as a new treatment option. This paper focuses on alternative treatment approaches, which are currently in various stages of development and can be divided into four therapeutic strategies. Direct killing of C. difficile (i) includes beside established antibiotics, less studied bacteriophages, and their derivatives, such as endolysins and tailocins. Restoration of microbiota composition and function (ii) is achieved with fecal microbiota transplantation, which has recently been approved, with standardized defined microbial mixtures, and with probiotics, which have been administered with moderate success. Prevention of deleterious effects of antibiotics on microbiota is achieved with agents for the neutralization of antibiotics that act in the gut and are nearing regulatory approval. Neutralization of C. difficile toxins (iii) which are crucial virulence factors is achieved with antibodies/antibody fragments or alternative binding proteins. Of these, the monoclonal antibody bezlotoxumab is already in clinical use. Immunomodulation (iv) can help eliminate or prevent C. difficile infection by interfering with cytokine signaling. Small-molecule agents without bacteriolytic activity are usually selected by drug repurposing and can act via a variety of mechanisms. The multiple treatment options described in this article provide optimism for the future treatment of C. difficile infection.

Structural basis of trypsin inhibition and entomotoxicity of cospin, serine protease inhibitor involved in defense of Coprinopsis cinerea fruiting bodies.

Cospin (PIC1) from Coprinopsis cinerea is a serine protease inhibitor with biochemical properties similar to those of the previously characterized fungal serine protease inhibitors, cnispin from Clitocybe nebularis and LeSPI from Lentinus edodes, classified in the family I66 of the MEROPS protease inhibitor classification. In particular, it exhibits a highly specific inhibitory profile as a very strong inhibitor of trypsin with K(i) in the picomolar range. Determination of the crystal structure revealed that the protein has a ß-trefoil fold. Site-directed mutagenesis and mass spectrometry results have confirmed Arg-27 as the reactive binding site for trypsin inhibition. The loop containing Arg-27 is positioned between the ß2 and ß3 strands, distinguishing cospin from other ß-trefoil-fold serine protease inhibitors in which ß4-ß5 or ß5-ß6 loops are involved in protease inhibition. Biotoxicity assays of cospin on various model organisms revealed a strong and specific entomotoxic activity against Drosophila melanogaster. The inhibitory inactive R27N mutant was not entomotoxic, associating toxicity with inhibitory activity. Along with the abundance of cospin in fruiting bodies of C. cinerea and the lack of trypsin-like proteases in the C. cinerea genome, these results suggest that cospin and its homologs are effectors of a fungal defense mechanism against fungivorous insects that function by specific inhibition of serine proteases in the insect gut.

Bivalent carbohydrate binding is required for biological activity of Clitocybe nebularis lectin (CNL), the N,N'-diacetyllactosediamine (GalNAcß1-4GlcNAc, LacdiNAc)-specific lectin from basidiomycete C. nebularis.

Lectins are carbohydrate-binding proteins that exert their biological activity by binding to specific cell glycoreceptors. We have expressed CNL, a ricin B-like lectin from the basidiomycete Clitocybe nebularis in Escherichia coli. The recombinant lectin, rCNL, agglutinates human blood group A erythrocytes and is specific for the unique glycan N,N'-diacetyllactosediamine (GalNAcß1-4GlcNAc, LacdiNAc) as demonstrated by glycan microarray analysis. We here describe the crystal structures of rCNL in complex with lactose and LacdiNAc, defining its interactions with the sugars. CNL is a homodimeric lectin, each of whose monomers consist of a single ricin B lectin domain with its ß-trefoil fold and one carbohydrate-binding site. To study the mode of CNL action, a nonsugar-binding mutant and nondimerizing monovalent CNL mutants that retain carbohydrate-binding activity were prepared. rCNL and the mutants were examined for their biological activities against Jurkat human leukemic T cells and the hypersensitive nematode Caenorhabditis elegans mutant strain pmk-1. rCNL was toxic against both, although the mutants were inactive. Thus, the bivalent carbohydrate-binding property of homodimeric CNL is essential for its activity, providing one of the rare pieces of evidence that certain activities of lectins are associated with their multivalency.

Expression of a hepatitis A virus antigen in Lactococcus lactis and Escherichia coli and evaluation of its immunogenicity.

An epidemic shift in Hepatitis A virus (HAV) infection has been observed in recent years in rapidly developing countries, with increasing numbers of severe adult cases which has led to renewed interest in vaccination. Our approach in vaccine development uses recombinant expression of the highly immunogenic HAV antigen VP1-P2a in food-grade lactic acid bacterium Lactococcus lactis and in Escherichia coli. We used genetic constructs that enable nisin-controlled expression of the antigen in L. lactis in three different forms: (a) intracellularly, (b) on the bacterial surface and (c) on the bacterial surface fused with the fragment of the E. coli flagellin molecule that can act as a molecular adjuvant. Expression of the two surface forms of the antigen was achieved in L. lactis, and the resulting antigen-displaying bacteria were administered orally to mice. Half the animals in each of the two groups developed specific IgGs, with titers increasing over time and reaching 1:422 without flagellin and 1:320 with flagellin. A much higher titer 1:25,803 was observed with the parenterally administered antigen, which was purified from E. coli. With the latter, a significant mucosal IgA response was also observed. Despite significant titers, the IgGs elicited with oral or parenteral administration could not prevent HAV from infecting cells in a virus neutralization assay, suggesting that the antibodies cannot recognize viral surface epitopes. Nevertheless, orally administered HAV antigen expressed in L. lactis elicited significant systemic humoral immune response showing the feasibility for development of effective HAV vaccine for mucosal delivery.

Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells.

Almost all of the 200 or so approved biopharmaceuticals have been produced in one of three host systems: the bacterium Escherichia coli, yeasts (Saccharomyces cerevisiae, Pichia pastoris) and mammalian cells. We describe the most widely used methods for the expression of recombinant proteins in the cytoplasm or periplasm of E. coli, as well as strategies for secreting the product to the growth medium. Recombinant expression in E. coli influences the cell physiology and triggers a stress response, which has to be considered in process development. Increased expression of a functional protein can be achieved by optimizing the gene, plasmid, host cell, and fermentation process. Relevant properties of two yeast expression systems, S. cerevisiae and P. pastoris, are summarized. Optimization of expression in S. cerevisiae has focused mainly on increasing the secretion, which is otherwise limiting. P. pastoris was recently approved as a host for biopharmaceutical production for the first time. It enables high-level protein production and secretion. Additionally, genetic engineering has resulted in its ability to produce recombinant proteins with humanized glycosylation patterns. Several mammalian cell lines of either rodent or human origin are also used in biopharmaceutical production. Optimization of their expression has focused on clonal selection, interference with epigenetic factors and genetic engineering. Systemic optimization approaches are applied to all cell expression systems. They feature parallel high-throughput techniques, such as DNA microarray, next-generation sequencing and proteomics, and enable simultaneous monitoring of multiple parameters. Systemic approaches, together with technological advances such as disposable bioreactors and microbioreactors, are expected to lead to increased quality and quantity of biopharmaceuticals, as well as to reduced product development times.

HWGMWSY, an unanticipated polystyrene binding peptide from random phage display libraries.

Phage display is a powerful technique for the discovery of peptide ligands that bind to various targets; however, ambiguous results often appear. Peptide HWGMWSY has been isolated repeatedly in our laboratory and by other research groups dealing with different protein and nonprotein targets, which led to a hypothesis that it may be a target-unrelated peptide interacting with polystyrene plastic surfaces. We compared binding properties and amplification rate of phage clone displaying the peptide HWGMWSY, a previously confirmed plastic binding clone WHWRLPS, and a control phage clone ASVQERK. An enzyme-linked immunosorbent assay and a phage elution assay confirmed that phage clone HWGMWSY binds to polystyrene. Surface plasmon resonance measurements on the other hand excluded the possibility of binding to bovine serum albumin, a common blocking agent in phage display experiments. Amplification rates of the above-noted phage clones were not statistically different. We therefore conclude that phage clone HWGMWSY was isolated in different selection procedures as a result of its affinity to polystyrene.

A complex of genes involved in adaptation of Leptinotarsa decemlineata larvae to induced potato defense.

The Colorado potato beetle (Leptinotarsa decemlineata) is the most important pest of potato in many areas of the world. One of the main reasons for its success lies in the ability of its larvae to counteract plant defense compounds. Larvae adapt to protease inhibitors (PIs) produced in potato leaves through substitution of inhibitor-sensitive digestive cysteine proteases with inhibitor-insensitive cysteine proteases. To get a broader insight into the basis of larval adaptation to plant defenses, we created a "suppression subtractive hybridisation" library using cDNA from the gut of L. decemlineata larvae fed methyl jasmonate-induced or uninduced potato leaves. Four hundred clones, randomly selected from the library, were screened for their relevance to adaptation with DNA microarray hybridizations. Selected enzyme systems of beetle digestion were further inspected for changes in gene expression using quantitative PCR and enzyme activity measurements. We identified two new groups of digestive cysteine proteases, intestains D and intestains E. Intestains D represent a group of structurally distinct digestive cysteine proteases, of which the tested members are strongly upregulated in response to induced plant defenses. Moreover, we found that other digestive enzymes also participate in adaptation, namely, cellulases, serine proteases, and an endopolygalacturonase. In addition, juvenile hormone binding protein-like (JHBP-like) genes were upregulated. All studied genes were expressed specifically in larval guts. In contrast to earlier studies that reported experiments based on PI-enriched artificial diets, our results increase understanding of insect adaptation under natural conditions.

Coupling CRISPR interference with FACS enrichment: New approach in glycoengineering of CHO cell lines for therapeutic glycoprotein production.

Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level.

CNL, a ricin B-like lectin from mushroom Clitocybe nebularis, induces maturation and activation of dendritic cells via the toll-like receptor 4 pathway.

A novel lectin, isolated from the basidiomycete mushroom Clitocybe nebularis and termed C. nebularis lectin (CNL), exhibits an immunostimulatory effect on the most potent antigen-presenting cells, the dendritic cells (DCs). Treatment of human monocyte-derived DCs with CNL in doses from 1 to 10 µg/ml resulted in a dose-dependent induction of overall DC maturation characteristics. Exposure of DCs to CNL for 48 hr resulted in extensive up-regulation of co-stimulatory molecules CD80 and CD86, as well as of the maturation marker CD83 and HLA-DR molecules. Such CNL-matured DCs (CNL-DCs) were capable of inducing a T helper type 1-polarized response in naive CD4+ CD45RA+ T cells in 5-day allogeneic co-cultures. The allostimulatory potential of CNL-DCs was significantly increased relative to untreated controls, as was their capacity to produce several pro-inflammatory cytokines such as interleukin-6, interleukin-8 and tumour necrosis factor-α. By using a specific Toll-like receptor 4 (TLR4) signalling inhibitor, CLI-095, as well as Myd88 inhibitory peptide, we have shown that DC activation by CNL is completely dependent on the TLR4 activation pathway. Furthermore, activation of TLR4 by CNL was confirmed via TLR4 reporter assay. Measurement of p65 nuclear factor-κB and p38 mitogen-activated protein kinase (MAPK) phosphorylation levels following CNL stimulation of DCs revealed primarily an increase in nuclear factor-κB activity, with less effect on the induction of p38 MAPK signalling than of lipopolysaccharide-matured DCs. The CNL had the ability to activate human DCs in such a way as to subsequently direct T helper type 1 T-cell responses. Our results encourage the use of mushroom-derived lectins for use in therapeutic strategies with aims such as to strengthen anti-tumour immune responses.

Identification of candidate carrier proteins for surface display on Lactococcus lactis by theoretical and experimental analyses of the surface proteome.

Lactococcus lactis is a lactic acid bacterium of proven safety for use in human oral applications. For this purpose, surface display of recombinant proteins is important, and new approaches for it are being sought. Analysis of the bacterial surface proteome is essential in identifying new candidate carrier proteins for surface display. We have made two different predictions of surface-associated proteins of L. lactis MG1363 by using Augur and LocateP software, which yielded 666 and 648 proteins, respectively. Surface proteins of L. lactis NZ9000, a derivative of MG1363, were identified by using a proteomics approach. The surface proteins were cleaved from intact bacteria, and the resulting peptides were identified by mass spectrometry. The latter approach yielded 80 proteins, 34 of which were not predicted by either software. Of the 80 proteins, 7 were selected for further study. These were cloned in frame with a C-terminal hexahistidine tag and overexpressed in L. lactis NZ9000 using nisin-controlled expression. Proteins of correct molecular weight carrying a hexahistidine tag were detected. Their surface localization was confirmed with flow cytometry. Basic membrane protein A (BmpA) was exposed at the highest level. To test BmpA as a candidate carrier protein, the hexahistidine tag was replaced by the B domain of staphylococcal protein A in the genetic construct. The B domain was displayed on the surface with BmpA as a carrier. The advantage of covalent BmpA binding was demonstrated. BmpA was thus shown to be a suitable candidate for a carrier protein in lactococcal surface display.