Understanding aspects of alginate biosynthesis and regulation by Pseudomonas aeruginosa : a thesis presented in partial fulfilment of the requirements of the degree of Doctor of Philosophy in Microbiology at Massey University, Palmerston North, New Zealand

Alginate is a medically and industrially important polymer produced by seaweeds and certain bacteria. The bacterium Pseudomonas aeruginosa over-produces alginate during cystic fibrosis lung infections, forming biofilms, making the infection difficult to treat. Bacteria make alginate using membrane spanning multi-protein complexes. Although alginate biosynthesis and regulation have been studied in detail, there are still major gaps in knowledge. In particular, the requirement of AlgL (a periplasmic alginate degrading enzyme) and role played by MucR (an inner membrane c-di-GMP modulator) are not well understood. Here I show that AlgL and MucR are not essential for alginate production during biofilm growth. My findings suggest that while catalytically active AlgL negatively affects alginate production, expressing catalytically inactive AlgL enhances alginate yields. Furthermore, preliminary data show AlgL is not required for the stability or functionality of the alginate biosynthesis complex, suggesting that it is a free periplasmic protein dispensable for alginate production. These findings support the prediction that the primary function of AlgL is to degrade misguided alginate from the periplasm. For MucR, I show for the first time that its sensor domain mediates nitrate-induced suppression of alginate biosynthesis. This appears to occur at multiple levels in a manner only partially dependent on c-di-GMP signaling. These results indicate that MucR is associated with the negative effect of nitrate (and possibly denitrification) on alginate production. On the basis of these results, I propose a combination of nitrate (or denitrification intermediates), exogenous lyases and antimicrobial agents could be used to eliminate established chronic biofilm infections. Furthermore, catalytically inactive AlgL and/or homologs of MucR with disabled sensor motifs could be harnessed in non-pathogenic bacteria for producing tailor-made alginates

Comparative study of encapsulated rhizome extract of Alpinia purpurata (Zingeberaceae) in alginate and alginate-chitosan

Encapsulation is a coating process of bioactive compound. Alpinia purpurata has been well known as lengkuas merah an Asian tropical herbal which contain phenylpropanoid, phenolic and flavonoid. Phenolic and flavonoid compounds is an agent that can be used as anti cancer. This research aim is to create a product of Alpinia purpurata extract which encapsulated in alginate or alginate-chitosan. Theproduct of encapsulated has been observed towards SEM ( Scanning Electron Microscophy) and spectrocophy Infra-Red method. Encapsulated product of lengkuas merah extract made through extrusion method in alginate and chitosan with ratio 1:1 (w/w) then dripped in 2% CaCl2.The Alpinia purpurata/alginate/chitosan microcapsules (APCAM) is better than alginate microcapsules (APAM)

A note on the effect of calcium alginate coating on quality of refrigerated strawberries.

peer-reviewedAn alginate-based edible coating was investigated for the preservation of the quality of strawberries during cold storage (5 °C). Strawberries were immersed, successively, in sodium alginate and calcium chloride solutions to generate a surface coating of calcium alginate. The quality of coated and non-coated strawberries was evaluated by weight loss, visible decay, titratable acidity, total soluble solids and reducing sugar concentration over a 14-day storage period. Results showed that coating with calcium alginate had no significant effects on weight loss or physicochemical parameters when compared to control fruit, but it did result in the postponement of visible decay during refrigerated storage

Alginate inhibits iron absorption from ferrous gluconate in a randomized controlled trial and reduces iron uptake into Caco-2 cells

Previous in vitro results indicated that alginate beads might be a useful vehicle for food iron fortification. A human study was undertaken to test the hypothesis that alginate enhances iron absorption. A randomised, single blinded, cross-over trial was carried out in which iron absorption was measured from serum iron appearance after a test meal. Overnight-fasted volunteers (n=15) were given a test meal of 200g cola-flavoured jelly plus 21 mg iron as ferrous gluconate, either in alginate beads mixed into the jelly or in a capsule. Iron absorption was lower from the alginate beads than from ferrous gluconate (8.5% and 12.6% respectively, p=0.003). Sub-group B (n=9) consumed the test meals together with 600 mg calcium to determine whether alginate modified the inhibitory effect of calcium. Calcium reduced iron absorption from ferrous gluconate by 51%, from 11.5% to 5.6% (p=0.014), and from alginate beads by 37%, from 8.3% to 5.2% (p=0.009). In vitro studies using Caco-2 cells were designed to explore the reasons for the difference between the previous in vitro findings and the human study; confirmed the inhibitory effect of alginate. Beads similar to those used in the human study were subjected to simulated gastrointestinal digestion, with and without cola jelly, and the digestate applied to Caco-2 cells. Both alginate and cola jelly significantly reduced iron uptake into the cells, by 34% (p=0.009) and 35% (p=0.003) respectively. The combination of cola jelly and calcium produced a very low ferritin response, 16.5% (p<0.001) of that observed with ferrous gluconate alone. The results of these studies demonstrate that alginate beads are not a useful delivery system for soluble salts of iron for the purpose of food fortification

A Preliminary Study on Using Multi-Nozzle Polymer Deposition System to Fabricate Composite Alginate/Carbon Nanotube Tissue Scaffolds

Three-dimensional composite alginate/single wall carbon nanotube (SWCNT) scaffolds encapsulated with endothelial cells were fabricated by a multi-nozzle biopolymer freeform deposition system. This system enables the converting of CAD designed scaffold pattern into process toolpaths and the use of computer control program to guide the nozzle deposition at spatial position for layered fabrication of 3D tissue scaffolds. The morphological, mechanical, structural and biological properties of as-fabricated scaffolds were characterized by optical microscope, SEM, Microtensile testing machine, Alamar Blue Assay, and Live-Dead Assay, respectively. The multi-nozzle deposition system demonstrated a highly efficient and effective process to build tissue scaffold or cell embedded constructs. Characterization results showed that the incorporation of SWCNT into alginate not only enhanced the mechanical strength of the scaffolds but also improved the cell affinity and the interaction with substrate. Further cell culture experimental results also showed that the incorporation of SWCNT in alginate enhanced endothelial cell proliferation compared with pure alginate scaffold.Mechanical Engineerin

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration

Antimicrobial and anti-inflammatory activity of chitosan-alginate nanoparticles: a targeted therapy for cutaneous pathogens.

Advances in nanotechnology have demonstrated potential application of nanoparticles (NPs) for effective and targeted drug delivery. Here we investigated the antimicrobial and immunological properties and the feasibility of using NPs to deliver antimicrobial agents to treat a cutaneous pathogen. NPs synthesized with chitosan and alginate demonstrated a direct antimicrobial activity in vitro against Propionibacterium acnes, the bacterium linked to the pathogenesis of acne. By electron microscopy (EM) imaging, chitosan-alginate NPs were found to induce the disruption of the P. acnes cell membrane, providing a mechanism for the bactericidal effect. The chitosan-alginate NPs also exhibited anti-inflammatory properties as they inhibited P. acnes-induced inflammatory cytokine production in human monocytes and keratinocytes. Furthermore, benzoyl peroxide (BP), a commonly used antiacne drug, was effectively encapsulated in the chitosan-alginate NPs and demonstrated superior antimicrobial activity against P. acnes compared with BP alone while demonstrating less toxicity to eukaryotic cells. Together, these data suggest the potential utility of topical delivery of chitosan-alginate NP-encapsulated drug therapy for the treatment of dermatologic conditions with infectious and inflammatory components

Daily ingestion of alginate reduces energy intake in free-living subjects

Sodium alginate is a seaweed-derived fibre that has previously been shown to moderate appetite in models of acute feeding. The mechanisms underlying this effect may include slowed gastric clearance and attenuated uptake from the small intestine. In order to assess whether alginate could be effective as a means of appetite control in free-living adults, 68 males and females (BMI range: 18.50-32.81 kgl M-2) completed this randomised, controlled two-way crossover intervention to compare the effects of 7 day daily ingestion of a strong-gelling sodium alginate formulation against a control. A sodium alginate with a high-guluronate content was chosen because, upon ingestion, it forms a strong gel in the presence of calcium ions. Daily preprandial ingestion of the sodium alginate formulation produced a significant 134.8 kcal (7%) reduction in mean daily energy intake. This reduced energy intake was underwritten by significant reductions in mean daily carbohydrate, sugar, fat, saturated fat and protein intakes. The absence of any significant interaction effects between the main effect of preload type and those of gender, BMI classification and/or timing of preload delivery indicates the efficacy of this treatment for individuals in different settings. These findings suggest a possible role for a strong-gelling sodium alginate formulation in the future management of overweight and obesity. (C) 2008 Elsevier Ltd. All rights reserved