ArnT proteins that catalyse the glycosylation of lipopolysaccharide share common features with bacterial N-oligosaccharyltransferases

ArnT is a glycosyltransferase that catalyses the addition of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A moiety of the lipopolysaccharide. This is a critical modification enabling bacteria to resist killing by antimicrobial peptides. ArnT is an integral inner membrane protein consisting of 13 predicted transmembrane helices and a large periplasmic C-terminal domain. We report here the identification of a functional motif with a canonical consensus sequence DEXRYAX(5)MX(3)GXWX(9)YFEKPX(4)W spanning the first periplasmic loop, which is highly conserved in all ArnT proteins examined. Site-directed mutagenesis demonstrated the contribution of this motif in ArnT function, suggesting that these proteins have a common mechanism. We also demonstrate that the Burkholderia cenocepacia and Salmonella enterica serovar Typhimurium ArnT C-terminal domain is required for polymyxin B resistance in vivo. Deletion of the C-terminal domain in B. cenocepacia ArnT resulted in a protein with significantly reduced in vitro binding to a lipid A fluorescent substrate and unable to catalyse lipid A modification with L-Ara4N. An in silico predicted structural model of ArnT strongly resembled the tertiary structure of Campylobacter lari PglB, a bacterial oligosaccharyltransferase involved in protein N-glycosylation. Therefore, distantly related oligosaccharyltransferases from ArnT and PglB families operating on lipid and polypeptide substrates, respectively, share unexpected structural similarity that could not be predicted from direct amino acid sequence comparisons. We propose that lipid A and protein glycosylation enzymes share a conserved catalytic mechanism despite their evolutionary divergence.

Cigarette smokers have exaggerated alveolar barrier disruption in response to lipopolysaccharide inhalation

RATIONALE: Cigarette smoke exposure is associated with an increased risk of the acute respiratory distress syndrome (ARDS); however, the mechanisms underlying this relationship remain largely unknown.

OBJECTIVE: To assess pathways of lung injury and inflammation in smokers and non-smokers with and without lipopolysaccharide (LPS) inhalation using established biomarkers.

METHODS: We measured plasma and bronchoalveolar lavage (BAL) biomarkers of inflammation and lung injury in smokers and non-smokers in two distinct cohorts of healthy volunteers, one unstimulated (n=20) and one undergoing 50 μg LPS inhalation (n=30).

MEASUREMENTS AND MAIN RESULTS: After LPS inhalation, cigarette smokers had increased alveolar-capillary membrane permeability as measured by BAL total protein, compared with non-smokers (median 274 vs 208 μg/mL, p=0.04). Smokers had exaggerated inflammation compared with non-smokers, with increased BAL interleukin-1β (p=0.002), neutrophils (p=0.02), plasma interleukin-8 (p=0.003), and plasma matrix metalloproteinase-8 (p=0.006). Alveolar epithelial injury after LPS was more severe in smokers than non-smokers, with increased plasma (p=0.04) and decreased BAL (p=0.02) surfactant protein D. Finally, smokers had decreased BAL vascular endothelial growth factor (VEGF) (p<0.0001) with increased soluble VEGF receptor-1 (p=0.0001).

CONCLUSIONS: Cigarette smoke exposure may predispose to ARDS through an abnormal response to a 'second hit,' with increased alveolar-capillary membrane permeability, exaggerated inflammation, increased epithelial injury and endothelial dysfunction. LPS inhalation may serve as a useful experimental model for evaluation of the acute pulmonary effects of existing and new tobacco products.

Lipopolysaccharide modification in Gram-negative bacteria during chronic infection

The Gram-negative bacterial lipopolysaccharide (LPS) is a major component of the outer membrane that plays a key role in host-pathogen interactions with the innate immune system. During infection, bacteria are exposed to a host environment that is typically dominated by inflammatory cells and soluble factors, including antibiotics, which provide cues about regulation of gene expression. Bacterial adaptive changes including modulation of LPS synthesis and structure are a conserved theme in infections, irrespective of the type or bacteria or the site of infection. In general, these changes result in immune system evasion, persisting inflammation, and increased antimicrobial resistance. Here, we review the modifications of LPS structure and biosynthetic pathways that occur upon adaptation of model opportunistic pathogens (Pseudomonas aeruginosa, Burkholderia cepacia complex bacteria, Helicobacter pylori and Salmonella enterica) to chronic infection in respiratory and gastrointestinal sites. We also discuss the molecular mechanisms of these variations and their role in the host-pathogen interaction.

Prevotella denticola Lipopolysaccharide from a Cystic Fibrosis Isolate Possesses a Unique Chemical Structure

We report the first complete structural characterization of the lipopolysaccharide (LPS) from a cystic fibrosis (CF) clinical isolate of Prevotella denticola (B003V1S1X). Chemical, spectroscopic, and spectrometric analyses revealed a unique rough-type LPS (LOS) structure. The structure has a highly negatively charged heptasaccharide core region containing hexoses, with the first two sugars, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and mannose, highly phosphorylated. Furthermore, the lipid A moiety has the typical structure for the genus Prevotella, and was also highly phosphorylated.

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

Febuxostat, an Inhibitor of Xanthine Oxidase, Suppresses Lipopolysaccharide-Induced MCP-1 Production via MAPK Phosphatase-1-Mediated Inactivation of JNK.

Excess reactive oxygen species (ROS) formation can trigger various pathological conditions such as inflammation, in which xanthine oxidase (XO) is one major enzymatic source of ROS. Although XO has been reported to play essential roles in inflammatory conditions, the molecular mechanisms underlying the involvement of XO in inflammatory pathways remain unclear. Febuxostat, a selective and potent inhibitor of XO, effectively inhibits not only the generation of uric acid but also the formation of ROS. In this study, therefore, we examined the effects of febuxostat on lipopolysaccharide (LPS)-mediated inflammatory responses. Here we show that febuxostat suppresses LPS-induced MCP-1 production and mRNA expression via activating MAPK phosphatase-1 (MKP-1) which, in turn, leads to dephosphorylation and inactivation of JNK in macrophages. Moreover, these effects of febuxostat are mediated by inhibiting XO-mediated intracellular ROS production. Taken together, our data suggest that XO mediates LPS-induced phosphorylation of JNK through ROS production and MKP-1 inactivation, leading to MCP-1 production in macrophages. These studies may bring new insights into the novel role of XO in regulating inflammatory process through MAPK phosphatase, and demonstrate the potential use of XO inhibitor in modulating the inflammatory processes.

Rôle du lipopolysaccharide dans la pathogenèse d'actinobacillus pleuropneumoniae et dans son interaction avec le système immunitaire inné

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal

Anti-lipopolysaccharide factor and crustin-III, the anti-white spot virus peptides in Penaeus monodon: Control of viral infection by up-regulation

White Spot Syndrome Virus (WSSV) is the most devastating disease affecting shrimp culture around the world. Though, considerable progress has been made in the detection and molecular characterization of WSSV in recent years, information pertaining to immune gene expression in shrimps with respect to WSSV infection remains limited. In this context, the present study was undertaken to understand the differential expression of antimicrobial peptide (AMP) genes in the haemocytes of Penaeus monodon in response to WSSV infection on a time-course basis employing semi-quantitative RT-PCR. The present work analyzes the expression profile of six AMP genes (ALF, crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5), eight WSSV genes (DNA polymerase, endonuclease, immediate early gene, latency related gene, protein kinase, ribonucleotide reductase, thymidine kinase and VP28) and three control genes (18S rRNA, β-actin and ELF) in P. monodon in response to WSSV challenge. Penaeidins were found to be up-regulated during early hours of infection and crustin-3 during late period of infection. However, ALF was found to be up-regulated early to late period of WSSV infection. The present study suggests that AMPs viz. ALF and crustin-3 play an important role in antiviral defense in shrimps. WSSV gene transcripts were detected post-challenge day 1 itself and increased considerably day 5 onwards. Evaluation of the control genes confirmed ELF as the most reliable control gene followed by 18S rRNA and β-actin for gene expression studies in shrimps. This study indicated the role of AMPs in the protection of shrimps against viral infection and their possible control through the up-regulation of AMPs

A new isoform of anti-lipopolysaccharide factor identified from the blue swimmer crab, Portunus pelagicus: Molecular characteristics and phylogeny

Anti-lipopolysaccharide factors are small proteins that bind and neutralize lipopolysaccharide and exhibit potent antimicrobial activities. This study presents the molecular characterization and phylogenetic analysis of the first ALF isoform (Pp-ALF1; JQ745295) identified from the hemocytes of Portunus pelagicus. The full length cDNA of Pp-ALF1 consisted of 880 base pairs encoding 293 amino acids with an ORF of 123 amino acids and contains a putative signal peptide of 24 amino acids. Pp-ALF1 possessed a predicted molecular weight (MW) of 13.86 kDa and theoretical isoelectric point (pI) of 8.49. Two highly conserved cysteine residues and putative LPS binding domain were observed in Pp-ALF1. Peptide model of Pp-ALF1 consisted of two α-helices crowded against a four-strand β-sheet. Comparison of amino acid sequences and neighbor joining tree showed that Pp-ALF1 has a maximum similarity (46%) to ALF present in Portunus trituberculatus followed by 39% similarity to ALF of Eriocheir sinensis and 38% similarity to ALFs of Scylla paramamosain and Scylla serrata. Pp-ALF1 is found to be a new isoform of ALF family and its characteristic similarity with other known ALFs signifies its role in protection against invading pathogens.

Two isoforms of anti-lipopolysaccharide factors identified and characterized from the hemocytes of portunid crabs, Portunus pelagicus and Scylla tranquebarica

Anti-lipopolysaccharide factors (ALFs), a type of cationic antimicrobial peptides (AMPs), and their derivatives are becoming predominant candidates for potential drugs in viral and bacterial diseases. This study reports the first ALF from the mud crab Scylla tranquebarica (StALF, JQ899453) and the second ALF isoform from the blue swimmer crab Portunus pelagicus (PpALF2, JQ899452). Both sequences encoded for precursor molecules, starting with a signal peptide containing 26 amino acid residues, followed by a highly cationic mature peptide, containing two conserved cysteine residues flanking a putative lipopolysaccharide (LPS)-binding domain. BLAST analysis revealed that both PpALF2 and StALF exhibited significant similarity with crustacean ALF sequences. The predicted molecular mass of the mature ALFs was 11.2 kDa with an estimated pI of 10.0. PpALF2 and StALF also showed the typical pattern of alternating hydrophobic and hydrophilic residues in their putative disulphide loop, suggesting that they comprise the same functional domain. Phylogenetic analysis showed that PpALF2 and StALF have similar evolutionary status and they were phylogenetically ancient immune effector molecules which may play an essential role in the host defense mechanism. The spatial structures of PpALF2 and StALF possessed four beta-strands and two alpha-helices. The results indicated that there were more than one ALF involved in crab immunity against various pathogens. ALFs would provide candidate promising therapeutic or prophylactic agents in health management and diseases control in crustacean aquaculture

Epidural analgesia with morphine or buprenorphine in ponies with lipopolysaccharide (LPS)-induced carpal synovitis

This study evaluated the analgesia effects of the epidural administration of 0.1 mg/kg bodyweight (BW) of morphine or 5 mu g/kg BW of buprenorphine in ponies with radiocarpal joint synovitis. Six ponies were submitted to 3 epidural treatments: the control group (C) received 0.15 mL/kg BW of a 0.9% sodium chloride (NaCl) solution; group M was administered 0.1 mg/kg BW of morphine; and group B was administered 5 mu g/kg BW of buprenorphine, both diluted in 0.9% NaCl to a total volume of 0.15 mL/kg BW administered epidurally at 10 s/mL. The synovitis model was induced by injecting 0.5 ng of lipopolysaccharide (LPS) in the left or right radiocarpal joint. An epidural catheter was later introduced in the lumbosacral space and advanced up to the thoracolumbar level. The treatment started 6 h after synovitis induction. Lameness, maximum angle of carpal flexion, heart rate, systolic arterial pressure, respiratory rate, temperature, and intestinal motility were evaluated before LPS injection (baseline), 6 h after LPS injection (time 0), and 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h after treatments. Although the model of synovitis produced clear clinical signs of inflammation, the lameness scores in group C were different from the baseline for only up to 12 h. Both morphine and buprenorphine showed a reduction in the degree of lameness starting at 0.5 and 6 h, respectively. Reduced intestinal motility was observed at 0.5 h in group M and at 0.5 to 1 h in group B. Epidural morphine was a more effective analgesic that lasted for more than 12 h and without side effects. It was concluded that morphine would be a valuable analgesic option to alleviate joint pain in the thoracic limbs in ponies.

Glucocorticoids Exacerbate Lipopolysaccharide-Induced Signaling in the Frontal Cortex and Hippocampus in a Dose-Dependent Manner

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established, evidence has accumulated showing that proinflammatory GC effects can occur in the brain, in a poorly understood manner. Using electrophoretic mobility shift assay, real-time PCR, and immunoblotting, we investigated the ability of varying concentrations of corticosterone (CORT, the GC of rats) to modulate lipopolysaccharide (LPS)-induced activation of NF-kappa B (nuclear factor kappa B), expression of anti- and proinflammatory factors and of the MAP (mitogen-activated protein) kinase family [ERK (extracellular signal-regulated kinase), p38, and JNK/ SAPK (c-Jun N-terminal protein kinase/ stress-activated protein kinase)], and AKT. In the frontal cortex, elevated CORT levels were proinflammatory, exacerbating LPS effects on NF-kappa B, MAP kinases, and proinflammatory gene expression. Milder proinflammatory GCs effects occurred in the hippocampus. In the absence of LPS, elevated CORT levels increased basal activation of ERK1/ 2, p38, SAPK/ JNK, and AKT in both regions. These findings suggest that GCs do not uniformly suppress neuroinflammation and can even enhance it at multiple levels in the pathway linking LPS exposure to inflammation.

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

Bone marrow chimeras and c-fms conditional ablation (mafia) mice reveal an essential role for resident myeloid cells in lipopolysaccharide/TLR4-induced corneal inflammation

The mammalian cornea contains an extensive network of resident macrophages and dendritic cells. To determine the role of these cells in LPS-induced corneal inflammation, TLR4−/− mice were sublethally irradiated and reconstituted with bone marrow cells from either enhanced GFP (eGFP)+/C57BL/6 or eGFP+/TLR4−/− mice. The corneal epithelium was abraded, LPS was added topically, and cellular infiltration to the corneal stroma and development of corneal haze were examined after 24 h. TLR4−/− mice reconstituted with C57BL/6, but not TLR4−/− bone marrow cells donor cells were found to cause infiltration of eGFP+ cells to the cornea, including neutrophils, and also increased corneal haze compared with saline-treated corneas. In a second experimental approach, corneas of transgenic macrophage Fas induced apoptosis (Mafia) mice were stimulated with LPS. These mice express eGFP and a suicide gene under control of the c-fms promoter, and systemic treatment with the FK506 dimerizer (AP20187) causes Fas-mediated apoptosis of monocytic cells. AP20187-treated mice had significantly fewer eGFP+ cells in the cornea than untreated mice. After stimulation with LPS neutrophil recruitment and development of corneal haze were impaired in AP20187-treated mice compared with untreated controls. Furthermore, LPS induced CXCL1/KC and IL-1α production within 4 h in corneas of untreated Mafia mice, which is before cellular infiltration; however, cytokine production was impaired after AP20187 treatment. Together, results from both experimental approaches demonstrate an essential role for resident corneal monocytic lineage cells (macrophages and dendritic cells) in development of corneal inflammation.