Pseudomonas aeruginosa Alters Peptidoglycan Composition under Nutrient Conditions Resembling Cystic Fibrosis Lung Infections.

Epidemic strains of Pseudomonas aeruginosa are highly virulent opportunistic pathogens with increased transmissibility and enhanced antimicrobial resistance. Understanding the cellular mechanisms behind this heightened virulence and resistance is critical. Peptidoglycan (PG) is an integral component of P. aeruginosa cells that is essential to its survival and a target for antimicrobials. Here, we examined the global PG composition of two P. aeruginosa epidemic strains, LESB58 and LESlike1, and compared them to the common laboratory strains PAO1 and PA14. We also examined changes in PG composition when the strains were cultured under nutrient conditions that resembled cystic fibrosis lung infections. We identified 448 unique muropeptides and provide the first evidence for stem peptides modified with O-methylation, meso-diaminopimelic acid (mDAP) deamination, and novel substitutions of mDAP residues within P. aeruginosa PG. Our results also present the first evidence for both d,l- and l,d-endopeptidase activity on the PG sacculus of a Gram-negative organism. The PG composition of the epidemic strains varied significantly when grown under conditions resembling cystic fibrosis (CF) lung infections, showing increases in O-methylated stem peptides and decreases in l,d-endopeptidase activity as well as an increased abundance of de-N-acetylated sugars and l,d-transpeptidase activity, which are related to bacterial virulence and antibiotic resistance, respectively. We also identified strain-specific changes where LESlike1 increased the addition of unique amino acids to the terminus of the stem peptide and LESB58 increased amidase activity. Overall, this study demonstrates that P. aeruginosa PG composition is primarily influenced by nutrient conditions that mimic the CF lung; however, inherent strain-to-strain differences also exist. IMPORTANCE Using peptidoglycomics to examine the global composition of the peptidoglycan (PG) allows insights into the enzymatic activity that functions on this important biopolymer. Changes within the PG structure have implications for numerous physiological processes, including virulence and antimicrobial resistance. The identification of highly unique PG modifications illustrates the complexity of this biopolymer in Pseudomonas aeruginosa. Analyzing the PG composition of clinical P. aeruginosa epidemic strains provides insights into the increased virulence and antimicrobial resistance of these difficult-to-eradicate infections.

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics.

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph-mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

Identifying lipids tightly bound to an integral membrane protein.

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear. Here, we use solid-state nuclear magnetic resonance (SSNMR) spectroscopy and mass spectrometry to characterize these molecules. Numerous correlations atypical for protein amino acids were found and assigned in the SSNMR spectra. The chemical shift patterns correspond to N-acetyl-d-glucosamine, N-acetyl-d-mannosaminuronic acid, and 4-acetamido-4,6-dideoxy-d-galactose which are part of the Enterobacterial Common Antigen (ECA). These sugars undergo rapid anisotropic motions and are likely linked flexibly to a rigid anchor that tightly binds ASR. Phosphorus NMR reveals several signals that are characteristic of monophosphates, further suggesting phosphatidylglyceride as the ECA lipid carrier which is anchored to ASR. In addition, NMR signals corresponding to common phospholipid phosphatidylethanolamine (PE) have been detected. The presence of PE tightly interacting with ASR was confirmed using liquid chromatography-mass spectrometry. This article commemorates Professor Michèle Auger and her contributions to membrane biophysics and Nuclear Magnetic Resonance.

Peptidoglycomics reveals compositional changes in peptidoglycan between biofilm- and planktonic-derived Pseudomonas aeruginosa.

Peptidoglycan (PG) is a critical component of the bacterial cell wall and is composed of a repeating ß-1,4-linked disaccharide of N-acetylglucosamine and N-acetylmuramic acid appended with a highly conserved stem peptide. In Gram-negative bacteria, PG is assembled in the cytoplasm and exported into the periplasm where it undergoes considerable maturation, modification, or degradation depending on the growth phase or presence of environmental stressors. These modifications serve important functions in diverse processes, including PG turnover, cell elongation/division, and antibiotic resistance. Conventional methods for analyzing PG composition are complex and time-consuming. We present here a streamlined MS-based method that combines differential analysis with statistical 1D annotation approaches to quantitatively compare PGs produced in planktonic- and biofilm-cultured Pseudomonas aeruginosa We identified a core assembly of PG that is present in high abundance and that does not significantly differ between the two growth states. We also identified an adaptive PG assembly that is present in smaller amounts and fluctuates considerably between growth states in response to physiological changes. Biofilm-derived adaptive PG exhibited significant changes compared with planktonic-derived PG, including amino acid substitutions of the stem peptide and modifications that indicate changes in the activity of amidases, deacetylases, and lytic transglycosylases. The results of this work also provide first evidence of de-N-acetylated muropeptides from P. aeruginosa The method developed here offers a robust and reproducible workflow for accurately determining PG composition in samples that can be used to assess global PG fluctuations in response to changing growth conditions or external stimuli.

A Botanical-Based Equine Nutraceutical Reduces Gastric Smooth Muscle Contractile Force In Vitro.

The objective of this study was to evaluate the effect of a botanical-based equine nutraceutical on contractility of gastric smooth muscle in vitro. Gastric ulcers are prevalent in performance horses and negatively impact horse welfare. Gastric hypermotility has been positively associated with the development of gastric ulceration in nonequine species, and reduction of hypermotility may be protective against their development. Stomachs from 12 pigs processed for food at a provincially inspected abattoir were collected within 1 hour of slaughter. Explants of nonglandular gastric tissue were prepared and suspended in a tissue bath, attached to a force transducer, in the presence or absence of a simulated digest extract of the nutraceutical. Tissue was stimulated to contract using increasing doses of acetylcholine. Peak and mean contractile force over 1 and 2 minutes after exposure to acetylcholine were measured. Exposure of gastric smooth muscle to the nutraceutical significantly reduced contractility of the tissue. These data provide support for the use of this nutraceutical to reduce contractility of nonglandular gastric smooth muscle and may indicate a protective effect of this nutraceutical in horses with mechanically induced gastric ulcers. Future studies are needed to clarify the role of gastric hypermotility on development of equine gastric ulcers and to determine the effect of this nutraceutical on equine gastric contractility and ulcerogenesis in vivo.

The synthesis of 16-androstene sulfoconjugates from primary porcine Leydig cell culture.

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

Multi-omics Integrative Investigation of Fatty Acid Metabolism in Obese and Lean Subcutaneous Tissue.

White adipose tissue (WAT) plays a central role in whole-body energy homeostasis through storage and release of fatty acids. A deeper understanding of the complex and highly integrated pathways regulating WAT fatty acid metabolism, and how they are altered with obesity, is necessary for diagnostic and therapeutic innovations in nutritional disorders. In this multi-omics study, we investigated the influence of obesity on fatty acid metabolism in human subcutaneous adipose tissue (SAT) using an approach that integrated transcriptomic, peptidomic, and fatty acid analyses. Notably, all analyses were conducted in the same adipose tissue sample from each participant, thus minimizing the chance of spurious results. In a sample of SAT from the periumbilical abdominal region of obese (n = 11, mean body mass index [BMI] = 35.0 ± 1.2 kg/m2) and lean subjects (n = 9, mean BMI = 22.1 ± 0.5 kg/m2), we found that obese SAT tended to have higher relative amounts of specific monounsaturated fatty acids and n-6 polyunsaturated fatty acids, and lower amounts of saturated fatty acids (p < 0.05). These changes were associated with differential regulation of lipogenic and lipolytic pathways in obese SAT. Fatty acid analysis showed changes in estimated fatty acid desaturase and elongase activities between lean and obese SAT (p < 0.05). Biomarkers of lipogenesis (e.g., fatty acid synthase protein) were differentially regulated between lean and obese SAT. These changes were noted in conjunction with increases in extracellular matrix remodeling proteins. Transcriptomic data revealed that the key regulators of lipolysis were reduced in obese SAT. This integrative multi-omics analysis collectively shows that obese SAT has a distinct fatty acid signature compared to lean SAT and the pathways underlying fatty acid metabolism are broadly regulated at the level of gene expression and protein abundance.

Structure and Mutational Analyses of Escherichia coli ZapD Reveal Charged Residues Involved in FtsZ Filament Bundling.

UNLABELLED: Bacterial cell division is an essential and highly coordinated process. It requires the polymerization of the tubulin homologue FtsZ to form a dynamic ring (Z-ring) at midcell. Z-ring formation relies on a group of FtsZ-associated proteins (Zap) for stability throughout the process of division. In Escherichia coli, there are currently five Zap proteins (ZapA through ZapE), of which four (ZapA, ZapB, ZapC, and ZapD) are small soluble proteins that act to bind and bundle FtsZ filaments. In particular, ZapD forms a functional dimer and interacts with the C-terminal tail of FtsZ, but little is known about its structure and mechanism of action. Here, we present the crystal structure of Escherichia coli ZapD and show it forms a symmetrical dimer with centrally located α-helices flanked by ß-sheet domains. Based on the structure of ZapD and its chemical cross-linking to FtsZ, we targeted nine charged ZapD residues for modification by site-directed mutagenesis. Using in vitro FtsZ sedimentation assays, we show that residues R56, R221, and R225 are important for bundling FtsZ filaments, while transmission electron microscopy revealed that altering these residues results in different FtsZ bundle morphology compared to those of filaments bundled with wild-type ZapD. ZapD residue R116 also showed altered FtsZ bundle morphology but levels of FtsZ bundling similar to that of wild-type ZapD. Together, these results reveal that ZapD residues R116, R221, and R225 likely participate in forming a positively charged binding pocket that is critical for bundling FtsZ filaments. IMPORTANCE: Z-ring assembly underpins the formation of the essential cell division complex known as the divisome and is required for recruitment of downstream cell division proteins. ZapD is one of several proteins in E. coli that associates with the Z-ring to promote FtsZ bundling and aids in the overall fitness of the division process. In the present study, we describe the dimeric structure of E. coli ZapD and identify residues that are critical for FtsZ bundling. Together, these results advance our understanding about the formation and dynamics of the Z-ring prior to bacterial cell division.

A vapourized Δ(9)-tetrahydrocannabinol (Δ(9)-THC) delivery system part I: development and validation of a pulmonary cannabinoid route of exposure for experimental pharmacology studies in rodents.

INTRODUCTION: Most studies evaluating the effects of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) in animal models administer it via a parenteral route (e.g., intraperitoneal (IP) or intravenous injection (IV)), however, the common route of administration for human users is pulmonary (e.g., smoking or vapourizing marijuana). A vapourized Δ(9)-THC delivery system for rodents was developed and used to compare the effects of pulmonary and parenteral Δ(9)-THC administration on blood cannabinoid levels and behaviour. METHODS: Sprague-Dawley rats were exposed to pulmonary Δ(9)-THC (1, 5, and 10mg of inhaled vapour) delivered via a Volcano® vapourizing device (Storz and Bickel, Germany) or to parenteral Δ(9)-THC (0.25, 0.5, 1.0, and 1.5mg/kg injected IP). Quantification of Δ(9)-THC and its psychoactive metabolite, 11-hydroxy-Δ(9)-THC (11-OH-Δ(9)-THC), in blood was determined by liquid chromatography/mass spectrometry (LC/MS). In order to verify the potential for the vapourization procedure to produce a robust conditioned place preference (CPP) or conditioned place avoidance CPA, classical conditioning procedures were systematically varied by altering the exposure time (10 or 20min) and number of exposed rats (1 or 2) while maintaining the same vapourization dose (10mg). RESULTS: Blood collected at 20min intervals showed similar dose-dependent and time-dependent changes in Δ(9)-THC and 11-OH-Δ(9)-THC for both pulmonary and parenteral administration of Δ(9)-THC. However, vapourized Δ(9)-THC induced CPP under certain conditions whereas IP-administered Δ(9)-THC induced CPA. DISCUSSION: These results support and extend the limited evidence (e.g., in humans, Naef et al., 2004; in rodents, Niyuhire et al., 2007) that Δ(9)-THC produces qualitatively different effects on behaviour depending upon the route of administration.

Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis.

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel ß-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.

A temporal examination of the planktonic and biofilm proteome of whole cell Pseudomonas aeruginosa PAO1 using quantitative mass spectrometry.

Chronic polymicrobial lung infections are the chief complication in patients with cystic fibrosis. The dominant pathogen in late-stage disease is Pseudomonas aeruginosa, which forms recalcitrant, structured communities known as biofilms. Many aspects of biofilm biology are poorly understood; consequently, effective treatment of these infections is limited, and cystic fibrosis remains fatal. Here we combined in-solution protein digestion of triplicate growth-matched samples with a high-performance mass spectrometry platform to provide the most comprehensive proteomic dataset known to date for whole cell P. aeruginosa PAO1 grown in biofilm cultures. Our analysis included protein-protein interaction networks and PseudoCAP functional information for unique and significantly modulated proteins at three different time points. Secondary analysis of a subgroup of proteins using extracted ion currents validated the spectral counting data of 1884 high-confidence proteins. In this paper we demonstrate a greater representation of proteins related to metabolism, DNA stability, and molecular activity in planktonically grown P. aeruginosa PAO1. In addition, several virulence-related proteins were increased during planktonic growth, including multiple proteins encoded by the pyoverdine locus, uncharacterized proteins with sequence similarity to mammalian cell entry protein, and a member of the hemagglutinin family of adhesins, HecA. Conversely, biofilm samples contained an uncharacterized protein with sequence similarity to an adhesion protein with self-association characteristics (AidA). Increased levels of several phenazine biosynthetic proteins, an uncharacterized protein with sequence similarity to a metallo-beta-lactamase, and lower levels of the drug target gyrA support the putative characteristics of in situ P. aeruginosa infections, including competitive fitness and antibiotic resistance. This quantitative whole cell approach advances the existing P. aeruginosa subproteomes and provides a framework for identifying and studying entire pathways critical to biofilm biology in this model pathogenic organism. The identification of novel protein targets could contribute to the development of much needed antimicrobial therapies to treat the chronic infections found in patients with cystic fibrosis.

Influence of O polysaccharides on biofilm development and outer membrane vesicle biogenesis in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is a common opportunistic human pathogen known for its ability to adapt to changes in its environment during the course of infection. These adaptations include changes in the expression of cell surface lipopolysaccharide (LPS), biofilm development, and the production of a protective extracellular exopolysaccharide matrix. Outer membrane vesicles (OMVs) have been identified as an important component of the extracellular matrix of P. aeruginosa biofilms and are thought to contribute to the development and fitness of these bacterial communities. The goal of this study was to examine the relationships between changes in the cell surface expression of LPS O polysaccharides, biofilm development, and OMV biogenesis in P. aeruginosa. We compared wild-type P. aeruginosa PAO1 with three chromosomal knockouts. These knockouts have deletions in the rmd, wbpM, and wbpL genes that produce changes in the expression of common polysaccharide antigen (CPA), O-specific antigen (OSA), or both. Our results demonstrate that changes in O polysaccharide expression do not significantly influence OMV production but do affect the size and protein content of OMVs derived from both CPA(-) and OSA(-) cells; these mutant cells also exhibited different physical properties from wild-type cells. We further examined biofilm growth of the mutants and determined that CPA(-) cells could not develop into robust biofilms and exhibit changes in cell morphology and biofilm matrix production. Together these results demonstrate the importance of O polysaccharide expression on P. aeruginosa OMV composition and highlight the significance of CPA expression in biofilm development.

Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment.

In many Gram-negative bacterial species, rough strains producing truncated lipopolysaccharide (LPS) generally exhibit defects in motility compared with smooth strains. However, the role that LPS plays in bacterial motility is not well understood. The goal of this study was to examine the relationship between LPS defects and motility of Pseudomonas aeruginosa. P. aeruginosa wild-type strain PAO1 and three isogenic mutants with defects in the rmlC, migA and wapR genes and producing truncated core oligosaccharide were investigated in terms of motility, attachment to glass and flagella expression. Compared with the wild-type, the three mutants showed significant retardation in both swarming motility on 0.5 % soft-agar plates and swimming motility on 0.3 % soft-agar plates. Moreover, attachment to abiotic surfaces was observed to be stronger in these mutants. The assembly of flagella appeared to be intact in these strains and the ability of individual cells to swim was unaffected. Flagellin proteins prepared from mutants rmlC and rmd, defective in the production of TDP-l-rhamnose and GDP-d-rhamnose, respectively, were compared and a change in molecular mass was observed only in the rmlC mutant. These data indicated that l-rhamnose, and not its enantiomer, d-rhamnose, is incorporated into the flagellin glycan of P. aeruginosa PAO1. The nucleotide-activated sugar precursor TDP-l-rhamnose is therefore shared between LPS biosynthesis and flagellin glycosylation in P. aeruginosa PAO1. Our results suggest that although biochemical precursors are shared by LPS and flagellin glycan biosynthesis, LPS truncations probably alter flagella-mediated motility in P. aeruginosa by modulating cell-surface attachment but not flagella synthesis.

Characterization of WbpB, WbpE, and WbpD and reconstitution of a pathway for the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid in Pseudomonas aeruginosa.

The lipopolysaccharide of Pseudomonas aeruginosa PAO1 contains an unusual sugar, 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid (d-ManNAc3NAcA). wbpB, wbpE, and wbpD are thought to encode oxidase, transaminase, and N-acetyltransferase enzymes. To characterize their functions, recombinant proteins were overexpressed and purified from heterologous hosts. Activities of His(6)-WbpB and His(6)-WbpE were detected only when both proteins were combined in the same reaction. Using a direct MALDI-TOF mass spectrometry approach, we identified ions that corresponded to the predicted products of WbpB (UDP-3-keto-d-GlcNAcA) and WbpE (UDP-d-GlcNAc3NA) in the coupled enzyme-substrate reaction. Additionally, in reactions involving WbpB, WbpE, and WbpD, an ion consistent with the expected product of WbpD (UDP-d-GlcNAc3NAcA) was identified. Preparative quantities of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA were enzymatically synthesized. These compounds were purified by high-performance liquid chromatography, and their structures were elucidated by NMR spectroscopy. This is the first report of the functional characterization of these proteins, and the enzymatic synthesis of UDP-d-GlcNAc3NA and UDP-d-GlcNAc3NAcA.

Altered protein expression in neutrophils of calves treated with dexamethasone.

The effect of glucocorticoid treatment on protein expression in bovine neutrophils was examined with a proteomic approach to address the mechanisms by which stress alters neutrophil function and predisposes to bacterial pneumonia in cattle. Calves 6 to 8 mo old were treated with dexamethasone (0.1 mg/kg), neutrophils were isolated 24 h later, and whole-cell lysates were examined by 2-dimensional electrophoresis. Differentially expressed protein spots were identified by peptide mass fingerprinting. The antimicrobial protein lactotransferrin was detected at increased amounts in the neutrophils of the dexamethasone-treated calves. Proteins detected at reduced amounts in the neutrophils of the dexamethasone-treated calves included annexin 1, phosphoglycerate mutase, Na(+) - K+ ATPase, and cathelicidin 1. These findings identify glucocorticoid-induced changes in the levels of neutrophil proteins involved in host defense, inflammation, and cellular metabolism and suggest additional mechanisms by which glucocorticoids affect neutrophil function.

Flagellin glycosylation in Pseudomonas aeruginosa PAK requires the O-antigen biosynthesis enzyme WbpO.

Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

Pilot study investigating the ability of an herbal composite to alleviate clinical signs of respiratory dysfunction in horses with recurrent airway obstruction.

Recurrent airway obstruction (RAO), known previously as chronic obstructive pulmonary disease (COPD), is a debilitating respiratory condition that significantly contributes to lost training days and illness in racehorses. Herbs are becoming increasingly popular for the prophylaxis or treatment of the clinical signs of RAO despite a paucity of research on efficacy and safety. We evaluated the ability of an herbal composite containing garlic, white horehound, boneset, aniseed, fennel, licorice, thyme, and hyssop to reduce the clinical signs of RAO, hypothesizing that the product would safely reduce signs and would improve the inflammatory cell profile within the lungs. The composite was fed to 6 horses with symptomatic RAO for 21 d in a crossover manner. Ventigraphs were used to record respiratory rate and intrapleural pressure; the proportion of inflammatory cells in fluid aspirated from the trachea was determined. Blood biochemical and hematologic screening was conducted to identify possible adverse effects. Treatment with the composite did not result in statistically significant changes in any of the parameters evaluated. A trend to a decrease in respiratory rate (P = 0.1) and an increase in the proportion of macrophages (P = 0.1) was observed in the horses receiving the herbal composite compared with placebo. These data indicate a potential for the herbal composite to safely reduce the elevated respiratory rate in horses with RAO. Future research with a greater number of horses is warranted to further characterize the effect of this product on horses with RAO.

Characterization of the major capsid proteins of myxoma virus particles using MALDI-TOF mass spectrometry.

The protein composition of poxvirus particles remains uncertain because of their large size and structural complexity. This has complicated the characterization of even well-studied Orthopoxviruses, like vaccinia virus, and little or nothing is known about the capsid composition of viruses belonging to other poxvirus genera. This paper describes methods that address this problem and have been used to identify 17 different Leporipoxvirus capsid proteins. Myxoma virus particles were purified using sucrose and Nicodenz gradient centrifugation and subfractionated into membrane and core fractions by thiol and detergent treatment. These materials were further fractionated using reverse-phase chromatography and SDS-PAGE and the resulting proteins identified by mass spectroscopy. Most of the myxoma proteins identified in this manner were homologs of either vaccinia virus structural proteins (F17R, L4R, J1R, H3L, A3L, A10L, A27L, and A45R) or virion-associated enzymes (I7L, H4L, D11L, A7L, and A22R). However, the myxoma homolog of the vaccinia P4a/A10L protein (M099L) differs from P4a protein in being proteolytically cleaved only once. M095L and M151R were also detected in core fractions. M095L and M151R are homologs of vaccinia A6L and B13R proteins, respectively, and poxvirus proteins not previously known to be capsid components. M093L, a protein of unknown function and having no certain Orthopoxvirus homolog associates with membrane fractions. These studies illustrate the conservation of Chordopoxvirion architecture and the methods that can be used to elucidate the proteins comprising these structures.

Evidence that WbpD is an N-acetyltransferase belonging to the hexapeptide acyltransferase superfamily and an important protein for O-antigen biosynthesis in Pseudomonas aeruginosa PAO1.

Di-N-acetylated uronic acid residues are unique sugar moieties observed in the lipopolysaccharides (LPS) of respiratory pathogens including several serotypes of Pseudomonas aeruginosa and several species of Bordetella. WbpD of P. aeruginosa PAO1 (serotype O5) is a putative 3-N-acetyltransferase that has been implicated in the biosynthesis of UDP-2,3-diacetamido-2,3-dideoxy-d-mannuronic acid [UDP-d-Man(2NAc3NAc)A], a precursor for the d-Man(2NAc3NAc)A residues in the B-band O antigen of this bacterium. A chromosomal knockout mutant of wbpD is incapable of producing either long-chain B-band O antigen (> or = 2 repeating units) or semi-rough LPS (lipid A-core + one repeat). Adding wbpD in trans restored LPS production to the wild-type level; this indicates that wbpD is important for biosynthesis of individual B-band O-antigen repeating units. WbpD contains left-handed beta-helical (LbetaH) structure as observed by Conserved Domain analysis and in silico secondary and tertiary structure predictions. This feature suggested that WbpD belongs to the hexapeptide acyltransferase (HexAT) superfamily of enzymes. WbpD was overexpressed as an N-terminally histidine-tagged fusion protein (His6-WbpD) and purified to > 95% purity. The protein was subjected to Far-UV circular dichroism spectroscopy, and the data revealed that WbpD contains left-handed helical structure, which substantiated in silico predictions made earlier. Results from SDS-PAGE, matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), and gel filtration analyses indicated that His6-WbpD has trimeric organization, consistent with the quaternary structure of HexATs. The binding of acetyl-CoA by WbpD was demonstrated by MALDI-TOF MS, suggesting that WbpD is an acetyltransferase that utilizes a direct-transfer reaction mechanism. Incubation of WbpD with acetyl-CoA significantly enhanced the stability of the protein and prevented precipitation over a course of 14 days. As a substrate for studying the enzymatic activity of WbpD is unavailable at present, a structure-based model for the LbetaH domain of WbpD was generated. Comparisons between this model and the LbetaH domains of known HexATs suggested that Lys136 plays a role in acetyl-CoA binding. A K136A site-directed mutant construct could only partially complement the wbpD knockout, and this mutation also reduced the stabilizing effects of acetyl-CoA, while a K136R mutation showed no discernible effect on complementation of the wbpD mutant or the stabilizing effects of acetyl-CoA on the purified mutant protein. A modified pathway was proposed for the biosynthesis of UDP-d-Man(2NAc3NAc)A, in which WbpD is involved in the catalysis of the fourth step by acting as a UDP-2-acetamido-3-amino-2,3-dideoxy-d-glucuronic acid 3-N-acetyltransferase.