Yeast extracts from different manufacturers and supplementation of amino acids and micro elements reveal a remarkable impact on alginate production by A. vinelandii ATCC9046.

BACKGROUND: In research and production, reproducibility is a key factor, to meet high quality and safety standards and maintain productivity. For microbial fermentations, complex substrates and media components are often used. The complex media components can vary in composition, depending on the lot and manufacturing process. These variations can have an immense impact on the results of biological cultivations. The aim of this work was to investigate and characterize the influence of the complex media component yeast extract on cultivations of Azotobacter vinelandii under microaerobic conditions. Under these conditions, the organism produces the biopolymer alginate. The focus of the investigation was on the respiration activity, cell growth and alginate production. RESULTS: Yeast extracts from 6 different manufacturers and 2 different lots from one manufacturer were evaluated. Significant differences on respiratory activity, growth and production were observed. Concentration variations of three different yeast extracts showed that the performance of poorly performing yeast extracts can be improved by simply increasing their concentration. On the other hand, the results with well-performing yeast extracts seem to reach a saturation, when their concentration is increased. Cultivations with poorly performing yeast extract were supplemented with grouped amino acids, single amino acids and micro elements. Beneficial results were obtained with the supplementation of copper sulphate, cysteine or a combination of both. Furthermore, a correlation between the accumulated oxygen transfer and the final viscosity (as a key performance indicator), was established. CONCLUSION: The choice of yeast extract is crucial for A. vinelandii cultivations, to maintain reproducibility and comparability between cultivations. The proper use of specific yeast extracts allows the cultivation results to be specifically optimised. In addition, supplements can be applied to modify and improve the properties of the alginate. The results only scratch the surface of the underlying mechanisms, as they are not providing explanations on a molecular level. However, the findings show the potential of optimising media containing yeast extract for alginate production with A. vinelandii, as well as the potential of targeted supplementation of the media.

Cation complexation by mucoid Pseudomonas aeruginosa extracellular polysaccharide.

Mucoid Pseudomonas aeruginosa is a prevalent cystic fibrosis (CF) lung colonizer, producing an extracellular matrix (ECM) composed predominantly of the extracellular polysaccharide (EPS) alginate. The ECM limits antimicrobial penetration and, consequently, CF sufferers are prone to chronic mucoid P. aeruginosa lung infections. Interactions between cations with elevated concentrations in the CF lung and the anionic EPS, enhance the structural rigidity of the biofilm and exacerbates virulence. In this work, two large mucoid P. aeruginosa EPS models, based on ß-D-mannuronate (M) and ß-D-mannuronate-α-L-guluronate systems (M-G), and encompassing thermodynamically stable acetylation configurations-a structural motif unique to mucoid P. aeruginosa-were created. Using highly accurate first principles calculations, stable coordination environments adopted by the cations have been identified and thermodynamic stability quantified. These models show the weak cross-linking capability of Na+ and Mg2+ ions relative to Ca2+ ions and indicate a preference for cation binding within M-G blocks due to the smaller torsional rearrangements needed to reveal stable binding sites. The geometry of the chelation site influences the stability of the resulting complexes more than electrostatic interactions, and the results show nuanced chemical insight into previous experimental observations.

Glycinebetaine: a versatile protectant to improve rice performance against aluminium stress by regulating aluminium uptake and translocation.

KEY MESSAGE: Glycinebetaine alleviates the detrimental effects of aluminium stress by regulating aluminium uptake and translocation, maintaining PSII activity, and activating the oxidative defence, thereby maintaining the growth and development of rice. Aluminium (Al) toxicity is one of the primary growth-limiting factors that limits plant growth and crop productivity in acidic soils. Rice (Oryza sativa L.) plants are susceptible to Al stress and do not naturally accumulate glycinebetaine (GB), one of the most effective protectants. Therefore, the objective of this study was to investigate whether exogenous GB can ameliorate the detrimental effects of Al stress on rice plants. Our results showed that the growth, development and biomass of rice were clearly inhibited under Al stress. However, exogenous GB application increased rice shoot growth and photosynthetic pigments contents, maintained photosystem II (PSII) activity, and activated the antioxidant defence system under Al stress. More importantly, GB may mediate the expression of Al uptake- and translocation-related genes, including OsALS1, OsNrat1, OsSTAR1 and OsSTAR2, and the galacturonic acid contents in rice roots under Al stress. Therefore, our findings highlight exogenous GB application is a valid approach to effectively combat Al toxicity by regulating physiological and biochemical processes in crops.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Extraction, purification, hypoglycemic and antioxidant activities of red clover (Trifolium pratense L.) polysaccharides.

Hot water extraction was applied to extract red clover (Trifolium pratense L.) polysaccharides (RCP) and the extraction conditions were optimized using the response surface methodology (RSM). An RCP yield of 12.72 ± 0.14% was achieved under the optimum extraction conditions: extracting time of 95 min, extracting temperature of 93 °C, and solvent-material ratio of 21 mL/g. A component named RCP-1.1 with the molecular weight of 7528.81 kDa was purified from RCP. RCP-1.1 was composed of glucose, galacturonic acid, arabinose, and galactose, with molar percentages of 52.54, 1.04, 16.31, and 30.11%, respectively. At the determination concentration of 10 mg/mL, the α-glucosidase inhibition ability of RCP-1.1 reached 86.72% of that of acarbose. The scavenging rates of RCP-1.1 (3.0 mg/mL) for DPPH and ABTS radicals reached 91.82% and 98.95% of that of ascorbic acid (3.0 mg/mL), respectively. Based on these results, RCP-1.1 possesses the potential to be used as a natural hypoglycemic agent or an antioxidant.

Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?

BACKGROUND: The heavy metal cadmium (Cd) accumulates in the environment due to anthropogenic influences. It is unessential and harmful to all life forms. The plant cell wall forms a physical barrier against environmental stress and changes in the cell wall structure have been observed upon Cd exposure. In the current study, changes in the cell wall composition and structure of Medicago sativa stems were investigated after long-term exposure to Cd. Liquid chromatography coupled to mass spectrometry (LC-MS) for quantitative protein analysis was complemented with targeted gene expression analysis and combined with analyses of the cell wall composition. RESULTS: Several proteins determining for the cell wall structure changed in abundance. Structural changes mainly appeared in the composition of pectic polysaccharides and data indicate an increased presence of xylogalacturonan in response to Cd. Although a higher abundance and enzymatic activity of pectin methylesterase was detected, the total pectin methylation was not affected. CONCLUSIONS: An increased abundance of xylogalacturonan might hinder Cd binding in the cell wall due to the methylation of its galacturonic acid backbone. Probably, the exclusion of Cd from the cell wall and apoplast limits the entry of the heavy metal into the symplast and is an important factor during tolerance acquisition.

Three-dimensional alginate hydrogels for radiobiological and metabolic studies of cancer cells.

The purpose of this study is to demonstrate calcium alginate hydrogels as a system for in vitro radiobiological and metabolic studies of cancer cells. Previous studies have established calcium alginate as a versatile three-dimensional (3D) culturing system capable of generating areas of oxygen heterogeneity and modeling metabolic changes in vitro. Here, through dosimetry, clonogenic and viability assays, and pimonidazole staining, we demonstrate that alginate can model radiobiological responses that monolayer cultures do not simulate. Notably, alginate hydrogels with radii greater than 500 µm demonstrate hypoxic cores, while smaller hydrogels do not. The size of this hypoxic region correlates with hydrogel size and improved cell survival following radiation therapy. Hydrogels can also be utilized in hyperpolarized magnetic resonance spectroscopy and extracellular flux analysis. Alginate therefore offers a reproducible, consistent, and low-cost means for 3D culture of cancer cells for radiobiological studies that simulates important in vivo parameters such as regional hypoxia and enables long-term culturing and in vitro metabolic studies.

Applying chlorogenic acid in an alginate scaffold of chondrocytes can improve the repair of damaged articular cartilage.

Damaged cartilage has very low regenerative potential which has led to the search for novel tissue-engineering approaches to help treat cartilage defects. While various approaches have been reported, there is no perfect treatment currently. In this study we evaluated the effects of a plant extract, chlorogenic acid (CGA), as part of chondrocyte transplantation on a model of knee joint injury in chicks. First, primary cultured chondrocytes used to evaluate the effects of CGA on chondrogenesis. Then using an articular cartilage injury model of chick knee we assessed the functional recovery after transplantation of the complexes containing chondrocytes and CGA in an alginate scaffold. Histological analysis, PCR, and western blot were further used to understand the underlying mechanisms. We showed that 60 µM CGA in alginate exhibited notable effects on stimulating chondrogenesis in vitro. Secondly, it was shown that the application of these complexes accelerated the recovery of injury-induced dysfunction by gait analysis when followed for 21 days. Histochemical analysis demonstrated that there was less abnormal vasculature formation, more chondrocyte proliferation and cartilage matrix synthesis in the presence of the complexes containing CGA. We discovered CGA treated transplantation up-regulated the expressions of Sox9 and Col2a1 which were responsible for the stimulation of chondrogenesis. Furthermore, the application of these complexes could suppress the abnormal angiogenesis and fibrosis at the injury site. Lastly, the elevated levels of inflammatory cytokines IL-1ß, TNF-α, p-p65, and MMPs expression were decreased in the presence of CGA. This may be caused through adjusting cellular redox homeostasis associated with Nrf2. This study suggests that combining chondrocytes and CGA on an alginate scaffold can improve the recovery of damaged articular cartilage.

Biphasic Scaffolds from Marine Collagens for Regeneration of Osteochondral Defects.

BACKGROUND: Collagens of marine origin are applied increasingly as alternatives to mammalian collagens in tissue engineering. The aim of the present study was to develop a biphasic scaffold from exclusively marine collagens supporting both osteogenic and chondrogenic differentiation and to find a suitable setup for in vitro chondrogenic and osteogenic differentiation of human mesenchymal stroma cells (hMSC). METHODS: Biphasic scaffolds from biomimetically mineralized salmon collagen and fibrillized jellyfish collagen were fabricated by joint freeze-drying and crosslinking. Different experiments were performed to analyze the influence of cell density and TGF-ß on osteogenic differentiation of the cells in the scaffolds. Gene expression analysis and analysis of cartilage extracellular matrix components were performed and activity of alkaline phosphatase was determined. Furthermore, histological sections of differentiated cells in the biphasic scaffolds were analyzed. RESULTS: Stable biphasic scaffolds from two different marine collagens were prepared. An in vitro setup for osteochondral differentiation was developed involving (1) different seeding densities in the phases; (2) additional application of alginate hydrogel in the chondral part; (3) pre-differentiation and sequential seeding of the scaffolds and (4) osteochondral medium. Spatially separated osteogenic and chondrogenic differentiation of hMSC was achieved in this setup, while osteochondral medium in combination with the biphasic scaffolds alone was not sufficient to reach this ambition. CONCLUSIONS: Biphasic, but monolithic scaffolds from exclusively marine collagens are suitable for the development of osteochondral constructs.

An Alginate/Cyclodextrin Spray Drying Matrix to Improve Shelf Life and Antioxidant Efficiency of a Blood Orange By-Product Extract Rich in Polyphenols: MMPs Inhibition and Antiglycation Activity in Dysmetabolic Diseases.

Alginate and ß-cyclodextrin were used to produce easily dosable and spray-dried microsystems of a dried blood orange extract with antidysmetabolic properties, obtained from a by-product fluid extract. The spray-dried applied conditions were able to obtain a concentrate dried extract without the loss of AOA and with TPC and TMA values of 35-40% higher than that of the starting material. They were also effective in producing microparticles with 80-100% of encapsulation efficiency. The 2% sodium alginate was capable of improving the extract shelf life, while the beta-cyclodextrin (1 : 1 molar ratio with dried extract) prolonged the extract antioxidant efficiency by 6 hours. The good inhibition effect of the dried extract on the AGE formation and the MMP-2 and MMP-9 activity is presumably due to a synergic effect exerted by both anthocyanin and bioflavonoid extract compounds and was improved by the use of alginate and cyclodextrin.

Binding mode of metal ions to the bacterial iron import protein EfeO.

The tripartite EfeUOB system functions as a low pH iron importer in Gram-negative bacteria. In the alginate-assimilating bacterium Sphingomonas sp. strain A1, an additional EfeO-like protein (Algp7) is encoded downstream of the efeUOB operon. Here we show the metal binding mode of Algp7, which carries a M_75 metallopeptidase motif. The Algp7 protein was purified from recombinant E. coli cells and was subsequently characterized using differential scanning fluorimetry, fluorescence spectrometry, atomic absorption spectroscopy, and X-ray crystallography. The fluorescence of a dye, SYPRO Orange, bound to denatured Algp7 in the absence and presence of metal ions was measured during heat treatment. The fluorescence profile of Algp7 in the presence of metals such as ferric, ferrous, and zinc ions, shifted to a higher temperature, suggesting that Algp7 binds these metal ions and that metal ion-bound Algp7 is more thermally stable than the ligand-free form. Algp7 was directly demonstrated to show an ability to bind copper ion by atomic absorption spectroscopy. Crystal structure of metal ion-bound Algp7 revealed that the metal ion is bound to the cleft surrounded by several acidic residues. Four residues, Glu79, Glu82, Asp96, and Glu178, distinct from the M_75 motif (His115xxGlu118), are coordinated to the metal ion. This is the first report to provide structural insights into metal binding by the bacterial EfeO element.

A solute-binding protein in the closed conformation induces ATP hydrolysis in a bacterial ATP-binding cassette transporter involved in the import of alginate.

The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.

Scaling up and scaling down the production of galactaric acid from pectin using Trichoderma reesei.

BACKGROUND: Bioconversion of D-galacturonic acid to galactaric (mucic) acid has previously been carried out in small scale (50-1000 mL) cultures, which produce tens of grams of galactaric acid. To obtain larger amounts of biologically produced galactaric acid, the process needed to be scaled up using a readily available technical substrate. Food grade pectin was selected as a readily available source of D-galacturonic acid for conversion to galactaric acid. RESULTS: We demonstrated that the process using Trichoderma reesei QM6a Δgar1 udh can be scaled up from 1 L to 10 and 250 L, replacing pure D-galacturonic acid with commercially available pectin. T. reesei produced 18 g L-1 galactaric acid from food-grade pectin (yield 1.00 g [g D-galacturonate consumed]-1) when grown at 1 L scale, 21 g L-1 galactaric acid (yield 1.11 g [g D-galacturonate consumed]-1) when grown at 10 L scale and 14 g L-1 galactaric acid (yield 0.77 g [g D-galacturonate consumed]-1) when grown at 250 L scale. Initial production rates were similar to those observed in 500 mL cultures with pure D-galacturonate as substrate. Approximately 2.8 kg galactaric acid was precipitated from the 250 L culture, representing a recovery of 77% of the galactaric acid in the supernatant. In addition to scaling up, we also demonstrated that the process could be scaled down to 4 mL for screening of production strains in 24-well plate format. Production of galactaric acid from pectin was assessed for three strains expressing uronate dehydrogenase under alternative promoters and up to 11 g L-1 galactaric acid were produced in the batch process. CONCLUSIONS: The process of producing galactaric acid by bioconversion with T. reesei was demonstrated to be equally efficient using pectin as it was with D-galacturonic acid. The 24-well plate batch process will be useful screening new constructs, but cannot replace process optimisation in bioreactors. Scaling up to 250 L demonstrated good reproducibility with the smaller scale but there was a loss in yield at 250 L which indicated that total biomass extraction and more efficient DSP would both be needed for a large scale process.

Screening of anionic-modified polymers in terms of stability, disintegration, and swelling behavior.

This study aimed to screen the stability, disintegration, and swelling behavior of chemically modified anionic polymers. Investigated polymers were well-known and widely used staples of the pharmaceutical and medical field, namely, alginate (AL), carboxymethyl cellulose (CMC), polycarbophil (PC), and hyaluronic acid (HA). On the basis of amide bond formation between the carboxylic acid moieties of anionic polymers and the primary amino group of the modification ligand cysteine (CYS), the modified polymers were obtained. Unmodified polymers served as controls throughout all studies. With the Ellman's assay, modification degrees were determined of synthesized polymeric excipients. Stability assay in terms of erosion study at physiological conditions were performed. Moreover, water uptake of compressed polymeric discs were evaluated and further disintegration studies according to the USP were carried out to define the potential ranking. Results ranking figured out PCCYS > CMCCYS > HACYS > ALCYS in terms of water uptake capacity compared to respective controls. Cell viability assays on Caco-2 cell line as well as on RPMI 2650 (ATTC CCL30) proved modification not being harmful to those. Due to the results of this study, an intense screening of prominent anionic polymer derivate was performed in order to help the pharmaceutical research for the best choice of polymeric excipients for developments of controlled drug release systems.

Isolation, Amino Acid Sequences, and Plausible Functions of the Galacturonic Acid-Binding Egg Lectin of the Sea Hare Aplysia kurodai.

Egg lectins occur in a variety of animals ranging from mollusks to vertebrates. A few examples of molluscan egg lectins have been reported, including that of the sea hare Aplysia kurodai; however, their biological functions in the egg remain unclarified. We report the isolation, determination of primary structure, and possible functions of A.kurodai lectin (AKL) from the egg mass of A. kurodai. We obtained AKL as an inseparable mixture of isoproteins with a relative molecular mass of approximately 32 kDa by affinity purification. The hemagglutinating activity of AKL against rabbit erythrocytes was inhibited most potently by galacturonic acid and moderately by xylose. Nucleotide sequencing of corresponding cDNA obtained by rapid amplification of cDNA ends (RACE) allowed us to deduce complete amino acid sequences. The mature polypeptides consisted of 218- or 219-amino acids with three repeated domains. The amino acid sequence had similarities to hypothetical proteins of Aplysia spp., or domain DUF3011 of uncharacterized bacterial proteins. AKL is the first member of the DUF3011 family whose function, carbohydrate recognition, was revealed. Treatment of the egg with galacturonic acid, an AKL sugar inhibitor, resulted in deformation of the veliger larvae, suggesting that AKL is involved in organogenesis in the developmental stage of A. kurodai.

Matrix exopolysaccharides; the sticky side of biofilm formation.

The Gram-negative pathogen Pseudomonas aeruginosa is found ubiquitously within the environment and is recognised as an opportunistic human pathogen that commonly infects burn wounds and immunocompromised individuals, or patients suffering from the autosomal recessive disorder cystic fibrosis (CF). During chronic infection, P. aeruginosa is thought to form structured aggregates known as biofilms characterised by a self-produced matrix which encases the bacteria, protecting them from antimicrobial attack and the host immune response. In many cases, antibiotics are ineffective at eradicating P. aeruginosa from chronically infected CF airways. Cyclic-di-GMP has been identified as a key regulator of biofilm formation; however, the way in which its effector proteins elicit a change in biofilm formation remains unclear. Identifying regulators of biofilm formation is a key theme of current research and understanding the factors that activate biofilm formation may help to expose potential new drug targets that slow the onset of chronic infection. This minireview outlines the contribution made by exopolysaccharides to biofilm formation, and describes the current understanding of biofilm regulation in P. aeruginosa with a particular focus on CF airway-associated infections.

Synthesis of cell composite alginate microfibers by microfluidics with the application potential of small diameter vascular grafts.

Fabrication of small diameter vascular grafts (SDVGs) with appropriate responses for clinical application is still challenging. In the present work, the production and characterization of solid alginate based microfibers as potential SDVG candidates through the method of microfluidics were considered original. A simple glass microfluidic device with a 'L-shape' cylindrical-flow channel in the microfluidic platform was developed. The gelation of microfibers occurred when the alginate solution and a CaCl2 solution were introduced as a core flow and as a sheath flow, respectively. The diameters of the microfibers could be controlled by varying the flow rates and the glass capillary tubes diameters at their tips. The generated microfibers had somewhat rough and porous surfaces, their suture retention strengths were comparable to the strength of other tissue engineered grafts. The encapsulated mesenchymal stem cells proliferated well in the microfibers, and showed a stable endothelialization under the angiogenesis effects of vascular endothelial growth factor and fibroblastic growth factor. The in vivo implant into the mice abdomens indicated that cell composite microfibers caused a mild host reaction. These encouraging results suggest great promise of the application of microfluidics as a future alternative in SDVGs engineering.

Alginate Particles with Ovalbumin (OVA) Peptide Can Serve as a Carrier and Adjuvant for Immune Therapy in B16-OVA Cancer Model.

BACKGROUND Alginate is a natural polysaccharide obtained from brown algae and has been shown to have numerous applications in biomedical science, such as wound healing, delivery of bioactive agents, and cell transplantation. Ovalbumin (OVA) peptide 323-339 has been reported to be involved in immune response.  MATERIAL AND METHODS This work investigated the use of alginate particles as a carrier and adjuvant for the immune therapy of cancer. Alginate particles loaded with OVA peptide were produced via emulsion. A tumor model was established in C57BL/6J mice via subcutaneous injection of 3×105 B16-OVA tumor cells. The effect of alginate/OVA peptide on cell viability was analyzed by use of the CCK-8 assay kit. Activation of macrophages was examined by checking cell surface makers CD40 and CD86 by FACs. RESULTS Alginate/OVA peptide inhibited tumor progression more effectively than using the peptide alone. The viability and uptake study illustrated that this particle is safe and non-toxic. The activation study demonstrated that alginate particles can promote the activation of surface markers on macrophages. ELISA assay showed that the particles with peptide can promote the secretion of inflammatory and effector cytokines from macrophages.  CONCLUSIONS This study demonstrated that alginate has dual functions in immune therapy of cancer, serving both as a carrier and an adjuvant.

OligoG CF-5/20 normalizes cystic fibrosis mucus by chelating calcium.

The goal of this study was to determine whether the guluronate (G) rich alginate OligoG CF-5/20 (OligoG) could detach cystic fibrosis (CF) mucus by calcium chelation, which is also required for normal mucin unfolding. Since bicarbonate secretion is impaired in CF, leading to insufficient mucin unfolding and thereby attached mucus, and since bicarbonate has the ability to bind calcium, we hypothesized that the calcium chelating property of OligoG would lead to detachment of CF mucus. Indeed, OligoG could compete with the N-terminus of the MUC2 mucin for calcium binding as shown by microscale thermophoresis. Further, effects on mucus thickness and attachment induced by OligoG and other alginate fractions of different length and composition were evaluated in explants of CF mouse ileum mounted in horizontal Ussing-type chambers. OligoG at 1.5% caused effective detachment of CF mucus and the most potent alginate fraction tested, the poly-G fraction of about 12 residues, had similar potency compared to OligoG whereas mannuronate-rich (M) polymers had minimal effect. In conclusion, OligoG binds calcium with appropriate affinity without any overt harmful effect on the tissue and can be exploited for treating mucus stagnation.

Activation Mechanism and Cellular Localization of Membrane-Anchored Alginate Polymerase in Pseudomonas aeruginosa.

The exopolysaccharide alginate, produced by the opportunistic human pathogen Pseudomonas aeruginosa, confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3', 5')-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization in vivo, an in silico structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution.IMPORTANCE The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen P. aeruginosa and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZAlg44 domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.