Heterogeneity of lipid A: comparison of lipid A types from different gram-negative bacteria

Chloroform-soluble purified lipid A preparations from 10 sources, including five Escherichia coli strains (EH100, K-12, O127, O111, RCDC), two Salmonella strains (Salmonella typhimurium, Salmonella minnesota R595), Shigella sonnei II, and a hybrid of Shigella flexneri and E. coli K-12, were compared with lipid A from S. flexneri. Purified lipid A from S. flexneri was earlier found to be composed of eight fractions. The various lipid A preparations were assayed by thin-layer chromatography. Chromatograms were stained for phosphate or carbohydrate by molybdenum blue or orcinol, respectively. The number of major bands found for each lipid A preparation varied between 7 and 10, depending on the source. Comparable bands, based on Rf, were found among all of the different lipid A preparations, but the quantity of each band varied between the sources of lipid A. Four bands (designated 2, 3, 7, and 8) were abundant in every preparation. Variations of conditions used for preparing lipid A, such as changing of hydrolysis time, did not affect the appearance of lipid A on thin-layer chromatography. Change in the type of acid used for hydrolysis also did not affect the band pattern, but it did change the quantitative amounts of the various bands to some degree.     

Serum amyloid A protein binds to outer membrane protein A of gram-negative bacteria

Serum amyloid A (SAA) is the major acute phase protein in man and most mammals. We observed SAA binding to a surprisingly large number of Gram-negative bacteria, including Escherichia coli, Salmonella typhimurium, Shigella flexneri, Klebsiella pneumoniae, Vibrio cholerae, and Pseudomonas aeruginosa. The binding was found to be high affinity and rapid. Importantly, this binding was not inhibited by high density lipoprotein with which SAA is normally complexed in serum. Binding was also observed when bacteria were offered serum containing SAA. Ligand blots following SDS-PAGE or two-dimensional gels revealed two major ligands of 29 and 35 kDa that bound SAA when probing with radiolabeled SAA or SAA and monoclonal anti-SAA. Following fractionation the ligand was found in the outer membrane fraction of E. coli and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to be outer membrane protein A (OmpA). OmpA-deficient E. coli did not bind SAA, and following purification of OmpA the protein retained binding activity. The ligands on other bacteria were likely to be homologues of OmpA because wild type, but not OprF-deficient, P. aeruginosa bound SAA.     

Polymorphic behavior of gram-negative bacteria membranes

Summary Freeze-fracture and ultrathin section electron microscopy as well as³¹P-NMR spectroscopy and light scattering ofEscherichia coli andPseudomonas putida cells under conditions promoting the ability of cells to take up exogenous DNA's (high concentrations of divalent cations and a specific temperature regime) reveal the extensive polymorphic changes and the formation of various structural defects in cellular membranes. Polymorphic changes occur during the heat shock at 42 to 44°C of the cells preincubated at 0°C in the presence of high concentration of Ca²⁺ or Ba²⁺ cations and include the formation of various vesicle- and tube-like structures, intermembrane and intercellular contacts followed by membrane fusion and sometimes even by cell fusion. The results obtained suggest the occurrence of phospholipid-enriched zones in the outer leaflet ofE. coli outer membrane. This suggestion is verified and confirmed with the help of phospholipase C, a specific phospholipid binding and digesting enzyme. The presented experimental evidence directly supports the suggestion of Ahkong et al. (Nature 253:194–195, 1975) on the identity of the mechanisms of membrane contact formation and membrane fusion in model and cellular membranes. The biological relevance of the polymorphic changes observed is shortly discussed.     

Molecular mechanisms of interaction of rabbit CAP18 with outer membranes of gram-negative bacteria.

The mechanism of interaction of the cationic antimicrobial protein (18 kDa), CAP18, with the outer membrane of Gram-negative bacteria was investigated applying transmission electron microscopy and voltage-clamp techniques on artificial planar bilayer membranes. Electron micrographs of bacterial cells exposed to CAP18 showed damage to the outer membrane of the sensitive Escherichia coli strains F515 and ATCC 11775, whereas the membrane of the resistant Proteus mirabilis strain R45 remained intact. Electrical measurements on various planar asymmetric bilayer membranes, one side consisting of a phospholipid mixture and the other of different phospholipids or of lipopolysaccharide (reconstitution model of the outer membrane), yielded information about the influence of CAP18 on membrane integrity. Addition of CAP18 to the side with the varying lipid composition led to lipid-specific adsorption of CAP18 and subsequent induction of current fluctuations due to the formation of transient membrane lesions at a lipid-specific clamp voltage. We propose that the applied clamp voltage leads to reorientation of CAP18 molecules adsorbed to the bilayer into an active transmembrane configuration, allowing the formation of lesions by multimeric clustering.     

A family of extracytoplasmic proteins that allow transport of large molecules across the outer membranes of gram-negative bacteria.

Seventeen fully sequenced and two partially sequenced extracytoplasmic proteins of purple, gram-negative bacteria constitute a homologous family termed the putative membrane fusion protein (MFP) family. Each such protein apparently functions in conjunction with a cytoplasmic membrane transporter of the ATP-binding cassette family, major facilitator superfamily, or heavy metal resistance/nodulation/cell division family to facilitate transport of proteins, peptides, drugs, or carbohydrates across the two membranes of the gram-negative bacterial cell envelope. Evidence suggests that at least some of these transport systems also function in conjunction with a distinct outer membrane protein. We report here that the phylogenies of these proteins correlate with the types of transport systems with which they function as well as with the natures of the substrates transported. Characterization of the MFPs with respect to secondary structure, average hydropathy, and average similarity provides circumstantial evidence as to how they may allow localized fusion of the two gram-negative bacterial cell membranes. The membrane fusion protein of simian virus 5 is shown to exhibit significant sequence similarity to representative bacterial MFPs.     

Lipopolysaccharide-protein complexes of the outer membrane of gram-negative bacteria

The evidence for occurring lipopolysaccharide-protein complexes in the outer membrane of gram-negative bacteria has been summarized. The composition and supramolecular structure of these complexes as well as their functions in microbial envelope and substantial role in membrane organization have been discussed. The biological properties of the complexes as endotoxins and O-specific antigens have been considered.     

Acyl-acyl carrier protein specificity of UDP-GlcNAc acyltransferases from gram-negative bacteria: relationship to lipid A structure.

Lipid A, the component of lipopolysaccharide that provides the membrane anchor of the core and O-antigen sugars, is known to contain characteristic R-3-hydroxy fatty acids bound to the 2,2' (N-linked) and 3,3' (O-linked) positions of the glucosamine disaccharide in different gram-negative bacteria. The studies reported here show that it is the acyl-acyl carrier protein specificities of the enzymes UDP-GlcNAc-O-acyltransferase and UDP-3-O-[(R)-3-hydroxyacyl]-GlcN-N-acyltransferase that determine the nature of these fatty acids.     

Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane

The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.     

Adaptive alterations in the outer membrane of gram-negative bacteria during human infection

The outer membrane of gram-negative bacteria is a dynamic structure that is capable of altering its ultrastructure and chemistry in order to adapt to changes in its environment. In human infections, outer-membrane alterations are known to play a role in mediating serum resistance, iron uptake, adaptation by Pseudomonas aeruginosa to colonization of the lungs of cystic fibrosis patients, and adaptive resistance to the polymyxin and aminoglycoside antibiotics. This adaptive antibiotic resistance is due to alterations in the cation binding sites within the outer membrane so that these cationic antibiotics can no longer penetrate through the membrane effectively. Adaptive resistance is not stable but is maintained only in the continued presence of the antibiotic. Hence, the role that this type of resistance to cationic antibiotics plays in clinical treatment of human infections remains inadequately assessed.     

Interactions of Synphilin-1 with phospholipids and lipid membranes

Synphilin-1 is an alpha-synuclein binding protein that is involved in the pathogenesis of Parkinson's disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610-640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson's disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function.     

A novel glycosphingolipid from gram-negative aquatic bacteria.

The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid. It was purified by two-stage silica gel-column chromatography. By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose. The ceramide portion consisted mainly (by 95% in the A. aquatica glycolipid and 80% in the F. major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine. The minor molecular species differed from the major one only in fatty acid structure. The glycolipid accounted for 8 and 11% of the total lipids extracted from A. aquatica NO-502 and F. major FM cells, respectively.     

ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria

The outer membrane of gram-negative bacteria is an asymmetric lipid bilayer with phospholipids and lipopolysaccharides (LPSs). β-Barreled outer membrane proteins and lipoproteins are embedded in the outer membrane. All of these constituents are essential to the function of the outer membrane. The transport systems for lipoproteins have been characterized in detail. An ATP-binding cassette (ABC) transporter, LolCDE, initiates sorting by mediating the detachment of lipoproteins from the inner membrane to form a water-soluble lipoprotein-LolA complex in the periplasm. Lipoproteins are then transferred to LolB at the outer membrane and are incorporated into the lipid bilayer. A model analogous to the Lol system has been suggested for the transport of LPS, where an ABC transporter, LptBFG, mediates the detachment of LPS from the inner membrane. Recent developments in the functional characterization of ABC transporters involved in the biogenesis of the outer membrane in gram-negative bacteria are discussed.     

革兰氏阴性菌外膜囊泡作为亚单位疫苗的研究进展北大核心CSCD

外膜囊泡(OMVs,Outer membrane vesicles)是一种在革兰氏阴性菌甚至某些革兰氏阳性菌中普遍存在的包含生物学活性物质的囊泡状结构,其大小在20–250 nm之间。其组成成分包括脂多糖、外膜蛋白、磷脂、DNA以及在形成过程中被外膜包裹的周质成分等。由于外膜囊泡不能复制且含有大量的细菌抗原,并能有效激活免疫系统,所以被认为是极具潜力的疫苗候选。虽然外膜囊泡从发现至今有50多年的历史,但针对其作为疫苗的潜力探究最近几年才开始,中国关于这方面的文献报道还很少。本文从外膜囊泡诱导免疫应答的机制以及其作为疫苗的研究进展两个方面概述了外膜囊泡可以作为一种新颖的防控疾病的疫苗策略,为今后外膜囊泡疫苗的深入研究提供参考。     

The outer membrane of gram-negative bacteria inhibits antibacterial activity of brochocin-C.

Brochocin-C is a two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754 that has a broad activity spectrum comparable to that of nisin. Brochocin-C has an inhibitory effect on EDTA-treated gram-negative bacteria, Salmonella enterica serovar Typhimurium lipopolysaccharide mutants, and spheroplasts of Typhimurium strains LT2 and SL3600. Brochocin-C treatment of cells and spheroplasts of strains of LT2 and SL3600 resulted in hydrolysis of ATP. The outer membrane of gram-negative bacteria protects the cytoplasmic membrane from the action of brochocin-C. It appears that brochocin-C is similar to nisin and possibly does not require a membrane receptor for its function; however, the difference in effect of the two bacteriocins on intracellular ATP indicates that they cause different pore sizes in the cytoplasmic membrane.