O139霍乱弧菌LPS基因在大肠杆菌中的克隆和表达

利用粘粒载体pCOS5构建了国内分离的O139霍乱弧菌的基因组文库,并从文库中筛选获得可以表达O139霍乱弧菌脂多糖的重组克隆株E.coliJM109(pMG310)。重组粘粒pMG310经酶切分析,所克隆的外源DNA片段大小为37kb。实验证明：重组克隆株E.coliJM109(pMG310)所表达的脂多糖具有良好的免疫原性及反应原性。     

细菌脂多糖诱导人单核细胞株对细菌脂蛋白的耐受性及其与肌动蛋白骨架关系

细菌脂多糖(LPS)可诱导宿主对LPS的耐受,但对细菌脂蛋白(BLP)是否存在交叉耐受,目前报道不一。采用人单核细胞株(THP-1),建立小剂量LPS诱导THP-1对LPS耐受的细胞模型;观察细胞肌动蛋白骨架、炎症因子TNF-α、IL-1β、IL-6的浓度及NF-κB的DNA结合活力的变化情况;探讨BLP交叉耐受及细胞骨架在其中的作用。结果显示,THP-1细胞经小剂量(10ng/ml)LPS、大剂量(100ng/ml)LPS或BLP刺激后,细胞形态严重变形,肌动蛋白重组,细胞周边肌动蛋白丝带消失,出现明显的肌动蛋白收缩团块及伪足,细胞核内NF-κB的DNA结合活性显著升高,培养上清液中炎症因子(TNF-α、IL-1β及IL-6)的释放显著增加;而小剂量LPS预刺激12h后,再用大剂量的LPS或BLP刺激6h,上述指标明显改善;采用细胞骨架肌动蛋白聚集破坏剂鬼笔环肽预处理后的THP-1细胞,可取消由小剂量LPS诱导的自身耐受及对BLP的交叉耐受;可见,细菌LPS、BLP(100ng/ml)可诱导THP-1细胞肌动蛋白骨架的改变,激活NF-κB信号通路,诱导炎性细胞因子TNF-α、IL-1、IL-6过度释放,激活宿主炎症细胞的炎症反应;而小剂量LPS预刺激后可诱导出THP-1细胞对LPS的自身耐受和对BLP的交叉耐受;细胞骨架肌动蛋白参与了小剂量LPS诱导THP-1细胞对LPS自身耐受和对BLP交叉耐受的形成。     

Lipid domains in bacterial membranes and the action of antimicrobial agents

There has been increasing interest in recent years in describing the lateral organization of membranes and the formation of membrane domains. Much of the focus in this area has been on the formation of cholesterol-rich domains in mammalian membranes. However, it is likely that there are domains in all biological membranes. One of the challenges has been to define the chemical composition, lifetime and size of these domains. There is evidence that bacteria have domains that are enriched in cardiolipin. In addition, the formation of lipid domains can be induced in bacteria by clustering negatively charged lipids with polycationic substances. Many antimicrobial compounds have multiple positive charges. Such polycationic compounds can sequester anionic lipids to induce lipid phase separation. The molecular interactions among lipids and their lateral packing density will be different in a domain from its environment. This will lead to phase boundary defects that will lower the permeability barrier between the cell and its surroundings. The formation of these clusters of anionic lipids may also alter the stability or composition of existing membrane domains that may affect bacterial function. Interestingly many antimicrobial agents are polycationic and therefore likely have some effect in promoting lipid phase segregation between anionic and zwitterionic lipids. However, this mechanism is expected to be most important for substances with sequential positive charges contained within a flexible molecule that can adapt to the arrangement of charged groups on the surface of the bacterial cell. When this mechanism is dominant it can allow the prediction of the bacterial species that will be most affected by the agent as a consequence of the nature of the lipid composition of the bacterial membrane.     

Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon

Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB₄ and LTC₄ synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB₄ and LTC₄. Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies – specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling – that may amplify inflammatory mediator production in atherosclerosis.     

Characterization of hydrogen peroxide production by Duox in bronchial epithelial cells exposed to Pseudomonas aeruginosa

Hydrogen peroxide production by the NADPH oxidase Duox1 occurs during activation of respiratory epithelial cells stimulated by purified bacterial ligands, such as lipopolysaccharide. Here, we characterize Duox activation using intact bacterial cells of several airway pathogens. We found that only Pseudomonas aeruginosa, not Burkholderia cepacia or Staphylococcus aureus, triggers H₂O₂ production in bronchial epithelial cells in a calcium-dependent but predominantly ATP-independent manner. Moreover, by comparing mutant Pseudomonas strains, we identify several virulence factors that participate in Duox activation, including the type-three secretion system. These data provide insight on Duox activation by mechanisms unique to P. aeruginosa.     

The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

Lipocalins, a widespread multifunctional family of small proteins (15-25kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K(d) range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K(d) range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.     

Nucleotide substrate recognition by UDP-N-acetylglucosamine acyltransferase (LpxA) in the first step of lipid A biosynthesis

Lipid A is an integral component of the lipopolysaccharide (LPS) that forms the selective and protective outer monolayer of Gram-negative bacteria, and is essential for bacterial growth and viability. UDP-N-acetylglucosamine acyltransferase (LpxA) initiates lipid A biosynthesis by catalyzing the transfer of R-3-hydroxymyristic acid from acyl carrier protein to the 3'-hydroxyl group of UDP-GlcNAc. The enzyme is a homotrimer, and previous studies suggested that the active site lies within a positively charged cleft formed at the subunit-subunit interface. The crystal structure of Escherichia coli LpxA in complex with UDP-GlcNAc reveals details of the substrate-binding site, with prominent hydrophilic interactions between highly conserved clusters of residues (Asn198, Glu200, Arg204 and Arg205) with UDP, and (Asp74, His125, His144 and Gln161) with the GlcNAc moiety. These interactions serve to bind and orient the substrate for catalysis. The crystallographic model supports previous results, which suggest that acylation occurs via nucleophilic attack of deprotonated UDP-GlcNAc on the acyl donor in a general base-catalyzed mechanism involving a catalytic dyad of His125 and Asp126. His125, the general base, interacts with the 3'-hydroxyl group of UDP-GlcNAc to generate the nucleophile. The Asp126 side-chain accepts a hydrogen bond from His125 and helps orient the general base to participate in catalysis. Comparisons with an LpxA:peptide inhibitor complex indicate that the peptide competes with both nucleotide and acyl carrier protein substrates.     

Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation

The regulatory NEMO (NF-κB essential modulator) protein has a crucial role in the canonical NF-κB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global ββα fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1β and TNF-α-induced NF-κB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-κB activation.     

Monomeric IgE and lipopolysaccharide synergistically prevent mast-cell apoptosis

The apoptosis of bone marrow-derived mast-cells (BMMCs) after growth factor withdrawal was significantly prevented by a high concentration of IgE in the absence of antigen, and further enhanced by the presence of Toll-like receptor4 (TLR4) ligand, lipopolysaccharide (LPS). The effect of LPS was mediated by TLR4, since TLR4-deficient BMMCs did not show synergistic effects with IgE. The neutralizing amount of anti-IL-3 did not reverse the anti-apoptotic effects of both IgE and combination with LPS. LPS treatment with monomeric IgE synergistically prevented the loss of mitochondrial membrane potentials and was associated with an enhanced expression of anti-apoptotic protein, Bcl-xL, or with a reduced expression of proapoptotic protein, Puma, and Bim, respectively. Altogether, these results suggest that LPS, in a TLR4-dependent manner, together with IgE, synergistically prevent mast-cell apoptosis and may contribute to regulate the tissue mast-cell number.     

ATP inhibits hydroxyl radical formation and the inflammatory response of stimulated whole blood even under circumstances of severe oxidative stress

We recently reported that Adenosine-5′-triphosphate (ATP) is able to inhibit the inflammatory reaction in stimulated whole blood. Many diseases, in which inflammatory reactions are involved, are associated with oxidative stress. In the present study, we therefore, investigated the effect of ATP on cytokine release in stimulated whole blood under conditions of oxidative stress, as simulated by pre-incubation of blood with hydrogen peroxide (H₂O₂). In the presence of H₂O₂, ATP at concentrations of 100 and 300 μM inhibited Tumour Necrosis factor-alpha (TNF-α) release and stimulated IL-10 release in LPS-PHA stimulated whole blood. Moreover, electron spin resonance (ESR) measurements showed that ATP and its breakdown product Adenosine-5′-diphosphate (ADP) attenuated spin trap-hydroxyl radical adduct formation in the Fenton reaction. Our results demonstrate that even in circumstances of severe oxidative stress, ATP has marked anti-inflammatory properties in stimulated whole blood. Moreover, the inhibition of the hydroxyl radical ESR signal indicates a direct attenuation of oxidative stress by ATP.