Characterization of the O-antigen Polymerase (Wzy) of Francisella tularensis

The O-antigen polymerase of Gram-negative bacteria has been difficult to characterize. Herein we report the biochemical and functional characterization of the protein product (Wzy) of the gene annotated as the putative O-antigen polymerase, which is located in the O-antigen biosynthetic locus of Francisella tularensis. In silico analysis (homology searching, hydropathy plotting, and codon usage assessment) strongly suggested that Wzy is an O-antigen polymerase whose function is to catalyze the addition of newly synthesized O-antigen repeating units to a glycolipid consisting of lipid A, inner core polysaccharide, and one repeating unit of the O-polysaccharide (O-PS). To characterize the function of the Wzy protein, a non-polar deletion mutant of wzy was generated by allelic replacement, and the banding pattern of O-PS was observed by immunoblot analysis of whole-cell lysates obtained by SDS-PAGE and stained with an O-PS-specific monoclonal antibody. These immunoblot analyses showed that O-PS of the wzy mutant expresses only one repeating unit of O-antigen. Further biochemical characterization of the subcellular fractions of the wzy mutant demonstrated that (as is characteristic of O-antigen polymerase mutants) the low molecular weight O-antigen accumulates in the periplasm of the mutant. Site-directed mutagenesis based on protein homology and topology, which was carried out to locate a catalytic residue of the protein, showed that modification of specific residues (Gly¹⁷⁶, Asp¹⁷⁷, Gly³²³, and Tyr³²⁴) leads to a loss of O-PS polymerization. Topology models indicate that these amino acids most likely lie in close proximity on the bacterial surface.     

Role of Helicobacter pylori rfaJ genes (HP0159 and HP1416) in lipopolysaccharide synthesis

The genome of Helicobacter pylori 26695 has been sequenced and the lipopolysaccharide (LPS) O sidechain of this strain has been shown to express both Lewis x and Lewis y units. To determine the role of HP0159 and HP1416, genes recognized as rfaJ homologs and implicated in LPS synthesis, isogenic mutants of H. pylori 26695 were generated. The LPS of mutant 26695::HP0159Kan did not express either Lewis epitope as detected by immunoblotting, whereas the control strain and 26695::HP1416Kan produced both epitopes. Structural analysis of the LPS of the mutants showed that HP0159 encodes an alpha(1,2/3)-glucosyltransferase whereas HP1416 encodes an alpha(1,2/4)-glucosyltransferase.     

Expression of T-cell immunoglobulin- and mucin-domain-containing molecules-1 and -3 (Tim-1 and Tim-3) in Helicobacter pylori infection

Background: Th immune response plays an important role in Helicobacter pylori (H. pylori) infection. Tim‐1 and Tim‐3 are expressed on terminally differentiated Th2 and Th1 cells, respectively, and participate in the regulation of Th immune response. Until now, the role of Tim in H. pylori infection remains unclear. Materials and Methods: (1) Lymphocytes isolated from the spleen of BALB/c mice were co‐cultured with different concentrations of viable H. pylori. Alternatively, mice were challenged by viable H. pylori to set up the H. pylori infection model. (2) The expression of Tim‐1 and Tim‐3 on mRNA level in lymphocytes or spleen of mice was determined by RT‐PCR. The percentage of Tim‐3‐positive cells was determined by flow cytometric analysis. The production of cytokine in supernatants was measured by standard sandwich cytokine ELISA. Results: (1) Co‐culture: At 12 hours, there was markedly decreased production of Tim‐1 and increased production of Tim‐3 in lymphocytes co‐cultured with H. pylori compared with normal control. The change of Th2 cytokine had the similar tendency as that of Tim‐1 expression; alternatively, the change of Th1 cytokine had the similar tendency as that of Tim‐3 expression. (2) Infection: Tim‐1 expression was declined in infected mice compared with control group; in the contrast, Tim‐3 expression was increased. Furthermore, the expression of Tim‐1 and Tim‐3 mRNA in spleen was significantly positively correlated with the level of Th2 and Th1 cytokine in gastric homogenized supernatant, respectively. Conclusion: H. pylori could inhibit the differentiation of T lymphocytes toward Th2 cells, promote the Th1 cell differentiation, and induce Th1‐biased immune response. The expression of Tim‐1 and Tim‐3 could reflect Th2 and Th1 immune response, respectively, which provide evidence for the prevention and treatment of H. pylori infection and correlation diseases through regulation of Tim‐1 and Tim‐3.     

The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)⁺. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.     

Reduction of Overall Helicobacter pylori Colonization Levels in the Stomach of Mongolian Gerbil by Lactobacillus johnsonii La1 (LC1) and Its in Vitro Activities against H. pylori Motility and Adherence

The effects of Lactobacillus johnsonii La1 (LC1) on Helicobacter pylori colonization in the stomach were investigated. H. pylori colonization and gastritis in LC1-inoculated Mongolian gerbils were significantly less intense than those in the control animals. LC1 culture supernatant (>10-kDa fraction) inhibited H. pylori motility and induced bacterial aggregation in human gastric epithelial cells, suggesting the potential of clinical use of LC1 product.     

Frame-shift mutations in NAD(P)H flavin oxidoreductase encoding gene (frxA) from metronidazole resistant Helicobacter pylori ATCC43504 and its involvement in metronidazole resistance

Metronidazole is a critical ingredient for combination therapies of Helicobacter pylori infection, the major cause of peptic ulcer and gastric cancer. It has been recently reported that metronidazole resistance from H. pylori ATCC43504 is caused by the insertion of a mini-IS605 sequence and deletion of sequences in an oxygen insensitive NAD(P)H nitroreductase encoding gene (rdxA). We also found that an additional gene (frxA) encoding NAD(P)H flavin oxidoreductase in the same strain was truncated by frame-shift mutations. To assess whether the frxA truncation is also involved in metronidazole resistance, metronidazole sensitive H. pylori strains ATCC43629 and SS1 were transformed by the truncated frxA gene cloned from strain ATCC43504. All transformed cells grew on agar plates containing 16 microg ml(-1) of metronidazole. The involvement of the frxA gene in metronidazole resistance was also confirmed by insertion inactivation of frxA and/or rdxA genes from strain ATCC43629 and one metronidazole sensitive clinical isolate H. pylori 2600. In addition, the frxA gene cloned from the H. pylori 2600 showed metronidazole nitroreductase activity in Escherichia coli and rendered ordinary metronidazole resistant E. coli to metronidazole sensitive cell. These results indicate that the frxA gene may also be involved in metronidazole resistance among clinical H. pylori isolates.     

Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes

Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance.     

Identification of a Bifunctional UDP-4-keto-pentose/UDP-xylose Synthase in the Plant Pathogenic Bacterium Ralstonia solanacearum Strain GMI1000, a Distinct Member of the 4,6-Dehydratase and Decarboxylase Family

The UDP-sugar interconverting enzymes involved in UDP-GlcA metabolism are well described in eukaryotes but less is known in prokaryotes. Here we identify and characterize a gene (RsU4kpxs) from Ralstonia solanacearum str. GMI1000, which encodes a dual function enzyme not previously described. One activity is to decarboxylate UDP-glucuronic acid to UDP-β-l-threo-pentopyranosyl-4″-ulose in the presence of NAD⁺. The second activity converts UDP-β-l-threo-pentopyranosyl-4″-ulose and NADH to UDP-xylose and NAD⁺, albeit at a lower rate. Our data also suggest that following decarboxylation, there is stereospecific protonation at the C5 pro-R position. The identification of the R. solanacearum enzyme enables us to propose that the ancestral enzyme of UDP-xylose synthase and UDP-apiose/UDP-xylose synthase was diverged to two distinct enzymatic activities in early bacteria. This separation gave rise to the current UDP-xylose synthase in animal, fungus, and plant as well as to the plant Uaxs and bacterial ArnA and U4kpxs homologs.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

The mechanical binding strengths of Helicobacter pylori BabA and SabA adhesins using an adhesion binding assay–ELISA,and its clinical relevance in Japan

To elucidate a potential role for H. pylori BabA and SabA adhesins in the pathogenesis of gastric mucosal lesions, the MBS of BabA and SabA was examined using an in‐house ABA‐ELISA. Ninety isolates from Japanese patients with gastric cancer (n= 43) and non‐cancerous (n= 47) lesions were subjected to an ABA‐ELISA which had been developed in‐house, and sequential analysis of the babA2 middle region. The BabA‐MBS was significantly higher in the cancer than the non‐cancer group (P= 0.019), but there was no significant difference for SabA‐MBS. A weak correlation between BabA‐MBS and SabA‐MBS (r= 0.418) was observed, the positive correlation being higher in the cancer than the non‐cancer group (r= 0.598 and 0.288, respectively). The isolates were classified into two groups: a BabA‐high‐binding and a BabA‐low‐binding group (in comparison to the average for BabA‐MBS). The average SabA‐MBS in the BabA‐high‐binding group was significantly higher than in the BabA‐low‐binding group (P < 0.0001). Analysis of babA2 middle region diversity (AD1–5) revealed that AD2‐type was predominant in isolates irrespective of BabA‐MBS. H. pylori BabA‐MBS might have an effect on SabA‐MBS and relate to the severity of gastric disorders, including gastric cancer. Evaluation of MBS of the combined two adhesins would be helpful for predicting damage in the H. pylori infected stomach.     

Disruption of proprotein convertase 1/3 (PC1/3) expression in mice causes innate immune defects and uncontrolled cytokine secretion

The proprotein convertase 1/3 is expressed in the regulated secretory pathway of neural and endocrine cells. Its major function is in the post-translational processing and activation of precursor proteins. The PC1/3 knock-out (KO) mouse model has allowed us to elucidate its physiological functions in studies focused primarily on neuroendocrine tissues. However, PC1/3 is also expressed in cells of the immune system, mainly in macrophages. The present study explores the effects of innate immune challenge in the PC1/3 KO mouse. PC1/3 KO mice have an enlarged spleen with marked disorganization of the marginal zone and red pulp. Immunohistochemical studies using various markers demonstrate a depletion of dendritic cells in PC1/3 KO spleens. When challenged with lipopolysaccharide, PC1/3 KO mice are more susceptible to septic shock than wild-type controls or other PC KO mice, such as PC2 and PC7 null mice. Plasma levels of proinflammatory cytokines (IL-6, IL-1β, and TNF-α) were very significantly elevated in PC1/3 KO mice, consistent with a hypercytokinemia, i.e. indicative of a major systemic uncontrolled inflammatory response or cytokine storm. Peritoneal macrophages isolated from PC1/3 KO mice also demonstrate elevated cytokine secretion when treated with LPS. Electron micrographs show morphological features indicating a prolonged activation of these cells following LPS stimulation. We also present evidence that the proinflammatory T(h)1 pathway is dominant in the PC1/3 KO mouse model. We conclude that aside from its important role in neuroendocrine functions PC1/3 also has an important role in the regulation of the innate immune system, most likely through the regulation of cytokine secretion in macrophages.     

高效液相色谱法同时测定银凤桃中的赤霉素和脱落酸

赤霉素(GA3)和脱落酸(ABA)是果实组织中的两个重要激素,本试验用SymmetryC18色谱柱(4.6mm×150mm),以乙腈和1.8%乙酸[V(CH3CN)∶V(1.8%CH3COOH)=1∶1]为流动相,流速为0.5mL·min-1,Wa-ters2487UV-检测器,在检测波长254nm,柱温25℃的条件下,同时分离并测定了银凤桃中的GA3和ABA。GA3和ABA的分离效果理想,回收率分别达到100.1%和99.8%,该方法测量灵敏度达10-2ng·g-1,精密度RSD%<0.1。     

A new beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Helicobacter pylori: Molecular cloning, enzymatic characterization, and structural modeling

Helicobacter pylori is a gram-negative pathogenic bacterium that causes peptic ulcer disease and gastric cancer, and studies of the related potent enzymes associated with this bacterium are urgent for the discovery of novel drug targets. In bacteria, beta-hydroxyacyl-acyl carrier protein (ACP) dehydratase (FabZ) is a potent enzyme in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxyacyl-ACP to trans-2-acyl-ACP. In this study, the cloning and enzymatic characterization of FabZ from H. pylori strain SS1 (HpFabZ) were reported, and the gene sequence of HpfabZ was deposited in the GenBank database. Enzyme dynamic analysis showed that HpFabZ had a K(m) of 82.6+/-4.3 microM toward its substrate analog crotonoyl-CoA. Dynamic light scattering and native-PAGE investigations suggested that HpFabZ exists as hexamer in native state. Enzymatic characterization and thermal-induced unfolding analysis based on circular dichroism spectral measurements indicated that HpFabZ is very stable against high temperature (90 degrees C). Such a high stability of HpFabZ was well elucidated by the strong H-bonds and hydrophobic interactions among the HpFabZ hexamer as investigated in the modeled HpFabZ hexamer structure. Our current study is hoped to provide useful information in better understanding the FabZ of H. pylori strain and further supply possible hints in the discovery of anti-bacterial compounds using HpFabZ as target.