Some lactic acid bacteria, especially
spp., possess adhesive properties enabling colonization of the human gastrointestinal tract. Two probiotic
strains, WCSF1 and 299v, display highly different ...mannose-specific adhesion, with
299v being superior to
WCFS1 based on a yeast agglutination assay. A straightforward correlation between the mannose adhesion capacity and domain composition of the mannose-specific adhesin (Msa) in the two strains has not been demonstrated previously. In this study, we analyzed the promoter regions upstream of the
gene encoding a mannose-specific adhesin in these two strains. The promoter region was mapped by primer extension and DNA sequence analysis, and only a single nucleotide change was identified between the two strains. However, Northern blot analysis showed a stronger
transcript band in 299v than in WCFS1 correlating with the different adhesion capacities. During the establishment of a high-throughput yeast agglutination assay, we isolated variants of WCFS1 that displayed a very strong mannose-specific adhesion phenotype. The region upstream of the
gene in these variants showed an inversion of a 104-bp fragment located between two perfectly inverted repeats present in the untranslated leader region. The inversion disrupts a strong hairpin structure that otherwise most likely would terminate the
transcript. In addition, the ribosome binding site upstream of the
gene, which is also masked within this hairpin structure, becomes accessible upon inversion, thereby increasing the frequency of translation initiation in the variant strains. Furthermore, Northern blot analysis showed a higher abundance of the
transcript in the variants than in the wild type, correlating with a strong-Msa phenotype.
Probiotic strains possess adhesive properties enabling colonization of the human intestinal tract through interactions between molecules present on the probiotic bacteria and components of the epithelial surface. In
, interaction is mediated through bacterial surface proteins like Msa, which binds to mannose residues present on the intestinal cells. Such interactions are believed to be important for the health-promoting effects of probiotics, including displacement of pathogens, immunomodulation, and protective effects on the intestinal barrier function. In this study, we have identified a new molecular switch controlling expression of the
gene in
strain WCFS1. Strains with increased
expression could be valuable in the development and manufacture of improved probiotic products.
Abstract
The use of the Gram-positive bacterium Lactococcus lactis in recombinant protein production has several advantages, including the organism's long history of safe use in food production and ...the fact that it does not produce endotoxins. Furthermore the current non-dairy L. lactis production strains contain few proteases and can secrete stable recombinant protein to the growth medium. The P170 expression system used for recombinant protein production in L. lactis utilizes an inducible promoter, P170, which is up-regulated as lactate accumulates in the growth medium. We have optimised the components of the expression system, including improved promoter strength, signal peptides and isolation of production strains with increased productivity. Recombinant proteins are produced in a growth medium with no animal-derived components as a simple batch fermentation requiring minimal process control. The accumulation of lactate in the growth medium does, however, inhibit growth and limits the yield from batch and fed-batch processes. We therefore combined the P170 expression system with the REED™ technology, which allows control of lactate concentration by electro-dialysis during fermentation. Using this combination, production of the Staphylococcus aureus nuclease reached 2.5 g L−1.
This study describes the development of a simple production system for manufacturing recombinant proteins and enzymes in a safe bacterial host.
Introduction
The risk of venous thromboembolism (VTE) is increased by more than 100-fold among hospitalised medical patients compared to subjects in the community. The Danish Council for the Use of ...Expensive Hospital Medicines has published national guidelines on thromboprophylaxis (TP) in which the risks of VTE and bleeding are balanced. We wanted to investigate the proportion of acutely admitted medical patients for whom thromboprophylaxis was indicated and to what extent the guidelines were followed.
Methods
Data from patients hospitalised at two medical wards were screened. We registered the proportion of patients for whom mechanical or pharmacologic TP (MTP and PTP, respectively) was indicated and whether national guidelines were followed. All data extraction and analyses were performed retrospectively.
Results
After exclusion criteria were applied, 340 cases remained. PTP was indicated in 26 patients (7.6%) but only 4 patients were treated besides 12 patients who were already in anticoagulant treatment at submission. Conversely, 8/306 patients, in whom TP was not indicated, were started on PTP. MTP was indicated in 8/340 patients (2.4%) but therapy was not initiated in any of them. The majority (320/340, 94.1%) of cases was managed in accordance with existing guidelines. However, this high proportion was mainly explained by the large number of untreated patients, where TP was not indicated.
Conclusion
A large proportion of hospitalised medical patients was managed in conflict with national guidelines. A systematic approach to TP in patients with acute medical illness should be implemented.
Plain Language Summary
Plain language summary available for this article.
In the present study, we used a proteomic approach to identify surface-associated proteins from the probiotic bacterium Lactobacillus plantarum 299v. Proteins were extracted from the cell surface ...using a mild wash in phosphate buffer and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Gel bands were excised and in-gel digested with trypsin. The resulting peptides were analysed by capillary-LC-ESI-MS/MS. The peptide sequences were used for a database search and allowed identification of a total of 29 proteins, many of which could potentially be involved in the action of probiotics in the gastrointestinal tract. The results provide the basis for future studies on the molecular mechanisms of probiotics.
Purpose
Production and characterization of a chimeric fusion protein (GMZ2’.10C) which combines epitopes of key malaria parasite antigens: glutamate-rich protein (GLURP), merozoite surface protein 3 ...(MSP3), and the highly disulphide bonded
Pf
s48/45 (10C). GMZ2’.10C is a potential candidate for a multi-stage malaria vaccine that targets both transmission and asexual life-cycle stages of the parasite.
Methods
GMZ2’.10C was produced in
Lactococcus lactis
and purified using either an immunoaffinity purification (IP) or a conventional purification (CP) method. Protein purity and stability was analysed by RP-HPLC, SEC-HPLC, 2-site ELISA, gel-electrophoresis and Western blotting. Structural characterization (mass analysis, peptide mapping and cysteine connectivity mapping) was performed by LC-MS/MS.
Results
CP-GMZ2’.10C resulted in similar purity, yield, structure and stability as compared to IP-GMZ2’.10C. CP-GMZ2’.10C and IP-GMZ2’.10C both elicited a high titer of transmission blocking (TB) antibodies in rodents. The intricate disulphide-bond connectivity of C-terminus
Pf
s48/45 was analysed by tandem mass spectrometry and was established for GMZ2’.10C and two reference fusion proteins encompassing similar parts of
Pf
s48/45.
Conclusion
GMZ2’.10C, combining GMZ2’ and correctly-folded
Pf
s48/45 can be produced by the
Lactoccus lactis
P170 based expression system in purity and quality for pharmaceutical development and elicit high level of TB antibodies. The cysteine connectivity for the 10C region of
Pf
s48/45 was revealed experimentally, providing an important guideline for employing the
Pf
s48/45 antigen in vaccine design.
Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as ...potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB 1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions, with an IC(50) value in the nanomolar range, is significantly more potent than the 3- and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3- and 4-BisPEB (IC(50), 1-2.5 μM). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.
Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, ...which may lead to unpredictable efficacy and/or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high-level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis.
A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N-terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2.
Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.
Secretion of β-1,3-glucanases by the arctic bacterial isolates 4221 and 4236, related to the genera Flavobacterium and Pedobacter, was discovered. Escherichia coli and Lactococcus lactis expression ...of β-1,3-glucanases Glc4221-1 and Glc4236-1 from the respective isolates was achieved. The enzymes hydrolyzed fungal cell walls and retained activity at low temperatures.
Walrus-tusk ivory and walrus-hide rope were highly desired goods in Viking Age north-west Europe. New finds of walrus bone and ivory in early Viking Age contexts in Iceland are concentrated in the ...south-west, and suggest extensive exploitation of nearby walrus for meat, hide and ivory during the first century of settlement. In Greenland, archaeofauna suggest a very different specialized long-distance hunting of the much larger walrus populations in the Disko Bay area that brought mainly ivory to the settlement areas and eventually to European markets. New lead isotopic analysis of archaeological walrus ivory and bone from Greenland and Iceland offers a tool for identifying possible source regions of walrus ivory during the early Middle Ages. This opens possibilities for assessing the development and relative importance of hunting grounds from the point of view of exported products.
Assessment of disease activity in inflammatory bowel disease (IBD), i.e., ulcerative colitis (UC) and Crohn's disease (CD), is done using clinical parameters and various biological disease markers. ...Ideally, a disease marker must: be able to identify individuals at risk of a given disorder, be disease specific, mirror the disease activity and, finally, be easily applicable for routine clinical purposes. However, no such disease markers have yet been identified for IBD. In this article, classical disease markers including erythrocyte sedimentation rate, acute phase proteins (especially orosomucoid and CRP), leukocyte and platelet counts, albumin, neopterin, and beta2-microglobulin will be reviewed together with emerging disease markers such as antibodies of the ANCA/ASCA type, cytokines (e.g., IL-1, IL-2Ralpha, IL-6, IL-8, TNF-alpha, and TNF-alpha receptors) and with various adhesion molecules. It is concluded that none of the pertinent laboratory surrogate markers of disease activity in IBD are specific or sensitive enough to replace basic clinical observation such as the number of daily bowel movements, general well-being, and other parameters in parallel. Further studies are highly warranted to identify and assess the clinical importance and applicability of new laboratory markers for the diagnosis or the disease activity of IBD.
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