Micromethods for the characterization of lipid A-core and O-antigen lipopolysaccharide

Methods for rapid and simple analysis of lipopolysaccharide (LPS) from bacterial whole-cell lysates or membrane preparations have contributed to advancing our knowledge of the genetics of the LPS biogenesis. LPS, a major constituent of the outer membranes in Gram-negative bacteria, has a complex mechanism of synthesis and assembly that requires the coordinated participation of many genes and gene products. This chapter describes a collection of methods routinely used in our laboratory for the characterization of LPS in Escherichia coli and other bacteria.

Two distinct but interchangeable mechanisms for flipping of lipid-linked oligosaccharides

Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.

Translocation of lipid-linked oligosaccharides across the ER membrane requires Rft1 protein

N-linked glycosylation of proteins in eukaryotic cells follows a highly conserved pathway. The tetradecasaccharide substrate (Glc3Man9GlcNAc2) is first assembled at the membrane of the endoplasmic reticulum (ER) as a dolichylpyrophosphate (Dol-PP)-linked intermediate, and then transferred to nascent polypeptide chains in the lumen of the ER. The assembly of the oligosaccharide starts on the cytoplasmic side of the ER membrane with the synthesis of a Man5GlcNAc2-PP-Dol intermediate. This lipid-linked intermediate is then translocated across the membrane so that the oligosaccharides face the lumen of the ER, where the biosynthesis of Glc3Man9GlcNAc2-PP-Dol continues to completion. The fully assembled oligosaccharide is transferred to selected asparagine residues of target proteins. The transmembrane movement of lipid-linked Man5GlcNAc2 oligosaccharide is of fundamental importance in this biosynthetic pathway, and similar processes involving phospholipids and glycolipids are essential in all types of cells. The process is predicted to be catalysed by proteins, termed flippases, which to date have remained elusive. Here we provide evidence that yeast RFT1 encodes an evolutionarily conserved protein required for the translocation of Man5GlcNAc2-PP-Dol from the cytoplasmic to the lumenal leaflet of the ER membrane.

Family-based association studies of lipid gene polymorphisms in coronary artery disease

Dysfunction of lipid-metabolizing proteins is implicated in the pathogenesis of coronary artery disease. Single nucleotide polymorphisms in genes that encode sterol regulatory binding protein-la, adenosine triphosphate binding cassette-A1, hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein were assessed as potential markers of disease susceptibility in a family-based study of 1,012 patients from 386 families. Association between single nucleotide polymorphisms and coronary artery disease was tested by the combined transmission disequilibrium test/sib transmission disequilibrium test and pedigree disequilibrium test. After Bonferroni's correction, the pedigree disequilibrium test demonstrated significant excess transmission (p < 0.0083) to affected patients of the hepatic lipase -514 T allele, which suggests that this may constitute a novel disease-susceptibility locus. (c) 2005 Elsevier Inc. All rights reserved.

Therapeutic potential of targeting lipid aldehydes and lipoxidation end-products in the treatment of ocular disease

Lipoxidation reactions and the subsequent accumulation of advanced lipoxidation end products (ALEs) have been implicated in the pathogenesis of many of the leading causes of visual impairment. Here, we begin by outlining some of the major lipid aldehydes produced through lipoxidation reactions, the ALEs formed upon their reaction with proteins, and the endogenous aldehyde metabolizing enzymes involved in protecting cells against lipoxidation mediated damage. Discussions are subsequently focused on the clinical and experimental evidence supporting the contribution of lipid aldehydes and ALEs in the development of ocular diseases. From these discussions, it is clear that inhibition of lipoxidation reactions and ALE formation could represent a new therapeutic avenue for the treatment of a broad range of ocular disorders. Current and emerging pharmacological strategies to prevent or neutralize the effects of lipid aldehydes and ALEs are therefore considered, with particular emphasis on the potential of these drugs for treatment of diseases of the eye.

Balancing Inflammatory, Lipid, and Xenobiotic Signaling Pathways by VSL#3, a Biotherapeutic Agent, in the Treatment of Inflammatory Bowel Disease

Background: The interleukin 10 knockout mouse (IL10-KO) is a model of human inflammatory bowel disease (IBD) used to Study host microbial interactions and the action of potential therapeutics. Using Affymetrix data analysis, important signaling pathways and transcription factors relevant to gut inflammation and antiinflammatory probiotics were identified.

Methods: Affymetrix microarray analysis on both wildtype (WT) and IL10-KO mice orally administered with and without the probiotic VSL#3 was performed and the results validated by real-time polymerase chain reaction (PCR), immunocytochemistry, proteomics, and histopathology. Changes in metabolically active bacteria were assessed with denaturing gradient gel electrophoresis (DGGE).

Results: Inflammation in IL10-KO mice was characterized by differential regulation of inflammatory, nuclear receptor, lipid, and xenobiotic signaling pathways. Probiotic intervention resulted in downregulation of CXCL9 (fold change [FC] = -3.98, false discovery rate [FDR] = 0.019), CXCL10 (FC = -4.83, FDR = 0.0008), CCL5 (FC -3.47 FDR = 0.017), T-cell activation (Itgal [FC = -4.72, FDR = 0.00009], Itgae [FC = -2.54 FDR = 0.0044]) and the autophagy gene IRGM (FC = -1.94, FDR = 0.01), a recently identified susceptibility gene in human IBD. Consistent with a marked reduction in integrins, probiotic treatment decreased the number of CCL5+ CD3+ double-positive T Cells and upregulated galectin2, which triggers apoptosis of activated T cells. Importantly, genes associated with lipid and PPAR signaling (PPAR alpha [FC = 2.36, FDR = 0.043], PPARGC1 alpha [FC 2.58, FDR = 0.016], Nrld2 [FC = 3.11, FDR = 0.0067]) were also upregulated. Altered microbial diversity was noted in probiotic-treated mice.

Conclusions: Bioinformatics analysis revealed important immune response. phagocytic and inflammatory pathways dominated by elevation of T-helper cell 1 type (TH1) transcription factors in IL10-KO mice. Probiotic intervention resulted in a site-specific reduction of these pathways but importantly upregulated PPAR, xenobiotic, and lipid signaling genes. potential antagonists of NF-kappa B inflammatory pathways.

Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A. Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

Role of lipid A acylation in Yersinia enterocolitica virulence

Yersinia enterocolitica is an important human pathogen. Y. enterocolitica must adapt to the host environment, and temperature is an important cue regulating the expression of most Yersinia virulence factors. Here, we report that Y. enterocolitica 8081 serotype O:8 synthesized tetra-acylated lipid A at 37 degrees C but that hexa-acylated lipid A predominated at 21 degrees C. By mass spectrometry and genetic methods, we have shown that the Y. enterocolitica msbB, htrB, and lpxP homologues encode the acyltransferases responsible for the addition of C(12), C(14) and C(16:1), respectively, to lipid A. The expression levels of the acyltransferases were temperature regulated. Levels of expression of msbB and lpxP were higher at 21 degrees C than at 37 degrees C, whereas the level of expression of htrB was higher at 37 degrees C. At 21 degrees C, an lpxP mutant was the strain most susceptible to polymyxin B, whereas at 37 degrees C, an htrB mutant was the most susceptible. We present evidence that the lipid A acylation status affects the expression of Yersinia virulence factors. Thus, expression of flhDC, the flagellar master regulatory operon, was downregulated in msbB and lpxP mutants, with a concomitant decrease in motility. Expression of the phospholipase yplA was also downregulated in both mutants. inv expression was downregulated in msbB and htrB mutants, and consistent with this finding, invasion of HeLa cells was diminished. However, the expression of rovA, the positive regulator of inv, was not affected in the mutants. The levels of pYV-encoded virulence factors Yops and YadA in the acyltransferase mutants were not affected. Finally, we show that only the htrB mutant was attenuated in vivo.

Pathogenic Yersinia enterocolitica strains increase the outer membrane permeability in response to environmental stimuli by modulating lipopolysaccharide fluidity and lipid A structure

Pathogenic biotypes of Yersinia enterocolitica (serotypes O:3, O:8, O:9, and O:13), but not environmental biotypes (serotypes O:5, O:6, O:7,8, and O:7,8,13,19), increased their permeability to hydrophobic probes when they were grown at pH 5.5 or in EGTA-supplemented (Ca(2+)-restricted) media at 37 degrees C. A similar observation was also made when representative strains of serotypes O:8 and O:5 were tested after brief contact with human monocytes. The increase in permeability was independent of the virulence plasmid. The role of lipopolysaccharide (LPS) in this phenomenon was examined by using Y. enterocolitica serotype O:8. LPS aggregates of bacteria grown in acidic or EGTA-supplemented broth took up more N-phenylnaphthylamine than LPS aggregates of bacteria grown in standard broth and also showed a marked increase in acyl chain fluidity which correlated with permeability, as determined by measurements obtained in the presence of hydrophobic dyes. No significant changes in O-antigen polymerization were observed, but lipid A acylation changed depending on the growth conditions. In standard medium at 37 degrees C, there were hexa-, penta-, and tetraacyl lipid A forms, and the pentaacyl form was dominant. The amount of tetraacyl lipid A increased in EGTA-supplemented and acidic media, and hexaacyl lipid A almost disappeared under the latter conditions. Our results suggest that pathogenic Y. enterocolitica strains modulate lipid A acylation coordinately with expression of virulence proteins, thus reducing LPS packing and increasing outer membrane permeability. The changes in permeability, LPS acyl chain fluidity, and lipid A acylation in pathogenic Y. enterocolitica strains approximate the characteristics in Yersinia pseudotuberculosis and Yersinia pestis and suggest that there is a common outer membrane pattern associated with pathogenicity.

Pomegranate polyphenols lower lipid peroxidation in adults with type 2 diabetes but have no effects in healthy volunteers:a pilot study

Aims. To examine the antioxidant and anti-inflammatory effects of pomegranate polyphenols in obese patients with type 2 diabetes (T2DM) (n = 8) and in healthy nondiabetic controls (n = 9). Methods. Participants received 2 capsules of pomegranate polyphenols (POMx, 1 capsule = 753?mg polyphenols) daily for 4 weeks. Blood draws and anthropometrics were performed at baseline and at 4 weeks of the study. Results. Pomegranate polyphenols in healthy controls and in T2DM patients did not significantly affect body weight and blood pressure, glucose and lipids. Among clinical safety profiles, serum electrolytes, renal function tests, and hematological profiles were not significantly affected by POMx supplementation. However, aspartate aminotransferase (AST) showed a significant increase in healthy controls, while alanine aminotransferase (ALT) was significantly decreased in T2DM patients at 4 weeks (P <0.05), though values remained within the normal ranges. Among the biomarkers of lipid oxidation and inflammation, oxidized LDL and serum C-reactive protein (CRP) did not differ at 4 weeks in either group, while pomegranate polyphenols significantly decreased malondialdehyde (MDA) and hydroxynonenal (HNE) only in the diabetic group versus baseline (P <0.05). Conclusions. POMx reduces lipid peroxidation in patients with T2DM, but with no effects in healthy controls, and specifically modulates liver enzymes in diabetic and nondiabetic subjects. Larger clinical trials are merited.

Low-energy cranberry juice decreases lipid oxidation and increases plasma antioxidant capacity in women with metabolic syndrome

Cranberries, high in polyphenols, have been associated with several cardiovascular health benefits, although limited clinical trials have been reported to validate these findings. We tested the hypothesis that commercially available low-energy cranberry juice (Ocean Spray Cranberries, Inc, Lakeville-Middleboro, Mass) will decrease surrogate risk factors of cardiovascular disease, such as lipid oxidation, inflammation, and dyslipidemia, in subjects with metabolic syndrome. In a randomized, double-blind, placebo-controlled trial, participants identified with metabolic syndrome (n = 15-16/group) were assigned to 1 of 2 groups: cranberry juice (480 mL/day) or placebo (480 mL/day) for 8 weeks. Anthropometrics, blood pressure measurements, dietary analyses, and fasting blood draws were conducted at screen and 8 weeks of the study. Cranberry juice significantly increased plasma antioxidant capacity (1.5 ± 0.6 to 2.2 ± 0.4 µmol/L [means ± SD], P <.05) and decreased oxidized low-density lipoprotein and malondialdehyde (120.4 ± 31.0 to 80.4 ± 34.6 U/L and 3.4 ± 1.1 to 1.7 ± 0.7 µmol/L, respectively [means ± SD], P <.05) at 8 weeks vs placebo. However, cranberry juice consumption caused no significant improvements in blood pressure, glucose and lipid profiles, C-reactive protein, and interleukin-6. No changes in these parameters were noted in the placebo group. In conclusion, low-energy cranberry juice (2 cups/day) significantly reduces lipid oxidation and increases plasma antioxidant capacity in women with metabolic syndrome.

The role of lipid peroxidation products and oxidative stress in activation of the canonical wingless-type MMTV integration site (WNT) pathway in a rat model of diabetic retinopathy

Our recent studies suggest that activation of the wingless-type MMTV integration site (WNT) pathway plays pathogenic roles in diabetic retinopathy and age-related macular degeneration. Here we investigated the causative role of oxidative stress in retinal WNT pathway activation in an experimental model of diabetes.

Green tea supplementation affects body weight, lipids, and lipid peroxidation in obese subjects with metabolic syndrome

To compare the effects of supplementation of green tea beverage or green tea extracts with controls on body weight, glucose and lipid profile, biomarkers of oxidative stress, and safety parameters in obese subjects with metabolic syndrome.

Freeze-dried strawberry powder improves lipid profile and lipid peroxidation in women with metabolic syndrome:baseline and post intervention effects

Strawberry flavonoids are potent antioxidants and anti-inflammatory agents that have been shown to reduce cardiovascular disease risk factors in prospective cohort studies. Effects of strawberry supplementation on metabolic risk factors have not been studied in obese populations. We tested the hypothesis that freeze-dried strawberry powder (FSP) will lower fasting lipids and biomarkers of oxidative stress and inflammation at four weeks compared to baseline. We also tested the tolerability and safety of FSP in subjects with metabolic syndrome. FSP is a concentrated source of polyphenolic flavonoids, fiber and phytosterols.