A complete lipopolysaccharide inner core oligosaccharide is required for resistance of Burkholderia cenocepacia to antimicrobial peptides and bacterial survival in vivo

Burkholderia cenocepacia is an important opportunistic pathogen of patients with cystic fibrosis. This bacterium is inherently resistant to a wide range of antimicrobial agents, including high concentrations of antimicrobial peptides. We hypothesized that the lipopolysaccharide (LPS) of B. cenocepacia is important for both virulence and resistance to antimicrobial peptides. We identified hldA and hldD genes in B. cenocepacia strain K56-2. These two genes encode enzymes involved in the modification of heptose sugars prior to their incorporation into the LPS core oligosaccharide. We constructed a mutant, SAL1, which was defective in expression of both hldA and hldD, and by performing complementation studies we confirmed that the functions encoded by both of these B. cenocepacia genes were needed for synthesis of a complete LPS core oligosaccharide. The LPS produced by SAL1 consisted of a short lipid A-core oligosaccharide and was devoid of O antigen. SAL1 was sensitive to the antimicrobial peptides polymyxin B, melittin, and human neutrophil peptide 1. In contrast, another B. cenocepacia mutant strain that produced complete lipid A-core oligosaccharide but lacked polymeric O antigen was not sensitive to polymyxin B or melittin. As determined by the rat agar bead model of lung infection, the SAL1 mutant had a survival defect in vivo since it could not be recovered from the lungs of infected rats 14 days postinfection. Together, these data show that the B. cenocepacia LPS inner core oligosaccharide is needed for in vitro resistance to three structurally unrelated antimicrobial peptides and for in vivo survival in a rat model of chronic lung infection.

Biosynthesis pathway of ADP-L-glycero-beta-D-manno-heptose in Escherichia coli

The steps involved in the biosynthesis of the ADP-L-glycero-beta-D-manno-heptose (ADP-L-beta-D-heptose) precursor of the inner core lipopolysaccharide (LPS) have not been completely elucidated. In this work, we have purified the enzymes involved in catalyzing the intermediate steps leading to the synthesis of ADP-D-beta-D-heptose and have biochemically characterized the reaction products by high-performance anion-exchange chromatography. We have also constructed a deletion in a novel gene, gmhB (formerly yaeD), which results in the formation of an altered LPS core. This mutation confirms that the GmhB protein is required for the formation of ADP-D-beta-D-heptose. Our results demonstrate that the synthesis of ADP-D-beta-D-heptose in Escherichia coli requires three proteins, GmhA (sedoheptulose 7-phosphate isomerase), HldE (bifunctional D-beta-D-heptose 7-phosphate kinase/D-beta-D-heptose 1-phosphate adenylyltransferase), and GmhB (D,D-heptose 1,7-bisphosphate phosphatase), as well as ATP and the ketose phosphate precursor sedoheptulose 7-phosphate. A previously characterized epimerase, formerly named WaaD (RfaD) and now renamed HldD, completes the pathway to form the ADP-L-beta-D-heptose precursor utilized in the assembly of inner core LPS.

Biosynthesis of inner core lipopolysaccharide in enteric bacteria identification and characterization of a conserved phosphoheptose isomerase

The lpcA locus has been identified in Escherichia coli K12 novobiocin-supersensitive mutants that produce a short lipopolysaccharide (LPS) core which lacks glyceromannoheptose and terminal hexoses. We have characterized lpcA as a single gene mapping around 5.3 min (246 kilobases) on the E. coli K12 chromosome and encoding a 22.6-kDa cytosolic protein. Recombinant plasmids containing only lpcA restored a complete core LPS in the E. coli strain chi711. We show that this strain has an IS5-mediated chromosomal deletion of 35 kilobases that eliminates lpcA. The LpcA protein showed discrete similarities with a family of aldose/ketose isomerases and other proteins of unknown function. The isomerization of sedoheptulose 7-phosphate, into a phosphosugar presumed to be D-glycero-D-mannoheptose 7-phosphate, was detected in enzyme reactions with cell extracts of E. coli lpcA+ and of lpcA mutants containing the recombinant lpcA gene. We concluded that LpcA is the phosphoheptose isomerase used in the first step of glyceromannoheptose synthesis. We also demonstrated that lpcA is conserved among enteric bacteria, all of which contain glyceromannoheptose in the inner core LPS, indicating that LpcA is an essential component in a conserved biosynthetic pathway of inner core LPS.

Modelling and Instrumentation of Stretch Blow Moulding

The injection stretch blow moulding process is used to manufacture PET containers used in the soft drinks and carbonated soft drinks industry. The process consists of a test tube like specimen known as a preform which is heated, stretch and blown into a mould to form the container. This research is focused on developing a validated simulation of the process thus enabling manufacturers to design their products in a virtual environment without the need to waste time, material and energy. The simulation has been developed using the commercial FEA package Abaqus and has been validated using state of the art data acquisition system consisting of measurements for preform temperature (inner and outer wall) using a device known as THERMOscan (Figure 1), stretch rod force and velocity, internal pressure and air temperature inside the preform using an instrumented stretch rod and the?exact?timing of when the preform touches the mould wall using contact sensors.? In addition, validation studies have also been performed by blowing a perform without a mould and using high sped imaging technology in cooperation with an advanced digital image correlation system (VIC 3D) to provided new quantitative information on the behaviour of PET during blowing.? The approach has resulted in a realistic simulation in terms of accurate input parameters, preform shape evolution and prediction of final properties.

The use of recycled demolition aggregate in precast concrete products – Phase III: Concrete pavement flags

A study undertaken at the University of Liverpool has investigated the potential for using construction and demolition waste (C&DW) derived aggregate in the manufacture of a range of precast concrete products, i.e. building and paving blocks and pavement flags. Phase III, which is reported here, investigated
concrete pavement flags. This was subsequent to studies on building and paving blocks. Recycled demolition aggregate can be used to replace newly quarried limestone aggregate, usually used in coarse (6 mm) and fine (4 mm-to-dust) gradings. The first objective was, as was the case with concrete building
and paving blocks, to replicate the process used by industry in fabricating concrete pavement flags in the laboratory. The ‘‘wet’’ casting technique used by industry for making concrete flags requires a very workable mix so that the concrete flows into the mould before it is compressed. Compression squeezes out water from the top as well as the bottom of the mould. This industrial casting procedure was successfully replicated in the laboratory by using an appropriately modified cube crushing machine and a special mould typical of what is used by industry. The mould could be filled outside of the cube crushing machine and then rolled onto a steel frame and into the machine for it to be compressed. The texture and mechanical properties of the laboratory concrete flags were found to be similar to the factory ones. The experimental work involved two main series of tests, i.e. concrete flags made with concrete- and
masonry-derived aggregate. Investigation of flexural strength was required for concrete paving flags. This is different from building blocks and paving blocks which required compressive and tensile splitting strength respectively. Upper levels of replacement with recycled demolition aggregate were determined
that produced similar flexural strength to paving flags made with newly quarried aggregates, without requiring an increase in the cement content. With up to 60% of the coarse or 40% of the fine fractions replaced with concrete-derived aggregates, the target mean flexural strength of 5.0 N/mm2 was still
achieved at the age of 28 days. There was similar detrimental effect by incorporating the fine masonry-derived aggregate. A replacement level of 70% for coarse was found to be satisfactory and also conservative. However, the fine fraction replacement could only be up to 30% and even reduced to 15% when used for mixes where 60% of the coarse fraction was also masonry-derived aggregate.