Detection of Shiga-like toxin on the outer membranes of Shigella species and Escherichia coli K-12

Abstract Outer membranes of Shigella species and E. coli K-12 carrying large invasive plasmids and isogenic non-invasive strains without plasmids were analyzed by SDS-PAGE. The immunoblotting analysis of the outer membrane proteins of these bacteria was performed with monoclonal antibody (mAb) made against A and B subunits of Shiga-like toxin (SLT). The SLT was detected in the outer membranes of S. dysenteriae 1 IDBM11, S. sonnei PNS20, S. flexneri M90T, S. dysenteriae 60R, and E. coli K-12 strain AB2463. The two other E. coli K-12 strains, C600 and 933J were included as controls for low and high toxin producers respectively. The outer membrane protein band of molecular weight 70 kDa was common to all bacterial strains studied. The most prominent band of 70 kDa protein was seen to be present in the high toxin producing plasmidless strain of S. dysenteriae 60R and the lysogenic strain of E. coli 933J. The invasive strains of S. dysenteriae 1 and S. flexneri M90T which carry the large invasive plasmids showed the least prominent band of 70 kDa protein.
The immunoblotting analysis of Shiga-toxin partially purified from the S. dysenteriae 60R strain revealed the absence of 70 kDa band on SDS-PAGE, instead the two dissociated subunits were seen. Furthermore, periplasmic Shiga-toxin proteins also showed the complete dissociation into A and B subunits. However, under the same denaturing conditions, the 70 kDa protein band cross-reacting with mAb against A and B subunits was still present in the outer membranes of all different strains.     

Actin Modulates the Gating of Neurospora crassa VDAC

VDAC forms the major pathway for metabolites across the mitochondrial outer membrane. The regulation of the gating of VDAC channels is an effective way to control the flow of metabolites into and out of mitochondria. Here we present evidence that actin can modulate the gating process of Neurospora crassa VDAC reconstituted into membranes made with phosphatidylcholine. An actin concentration as low as 50 nm caused the VDAC-mediated membrane conductance to drop by as much as 85% at elevated membrane potentials. Actin's effect could be quickly reversed by adding pronase to digest the protein. α-Actin, from mammalian muscle, has a stronger effect than β- and γ-actin from human platelets. The monomeric form of actin, G-actin, is effective. Stabilization of the fibrous form, F-actin, with the mushroom toxin, phalloidin, blocks the effect of actin on VDAC, indicating that F-actin might be ineffective. Cytochalasin B did not interfere with the ability of actin to favor VDAC closure. DNase-I did effectively block actin's effect on VDAC, and VDAC decreased actin's inhibitory effect on DNase-I activity, indicating that N. crassa VDAC competes with DNase-I for the same binding site on actin. The actin-VDAC interaction might be a mechanism by which actin regulates energy metabolism. Received: 28 August 2000/Revised: 1 December 2000     

Purification and characterization of outer membrane protein P6, a vaccine antigen of non-typeable Haemophilus influenzae

Outer membrane protein P6 is a promising vaccine antigen with potential to prevent infections caused by non-typeable Haemophilus influenzae. A convenient and reliable method for the purification of P6 and an assessment of the purity, yield, protein structure, antigenicity and immunogenicity of the purified protein are described. The method begins with intact H. influenzae and utilizes a series of incubations and centrifugations using a single buffer to remove all cell components with the exception of the peptidoglycan to which the P6 is associated. P6 is dissociated from the complex with heat and the insoluble peptidoglycan is removed by centrifugation. The procedure yields highly purified P6. Contamination with lipooligosaccharide is less than 0.025 endotoxin U per microgr P6. The yield of P6 is approximately 2 mg of P6 per l H. influenzae culture. The purified P6 retains both the secondary and tertiary structure as measured by circular dichroism and analysis with monoclonal antibodies. The purified P6 is immunogenic in animals. A convenient method for purifying P6 which retains antigenicity and immunogenicity will be an important tool for future studies of the vaccine potential of P6.     

A plasmid-based vector system for the cloning and expression of Helicobacter pylori genes encoding outer membrane proteins

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. We observed that the functional expression of such proteins is deleterious to Escherichia coli, the host bacterium used for gene cloning. Therefore, a general method was developed for the functional expression of such genes on a shuttle vector in H. pylori, which has been termed SOMPES (Shuttle vector-based Outer Membrane Protein Expression System). The intact, active gene is reconstituted by recombination in H. pylori from partial gene sequences cloned on an E. coli-H. pylori shuttle vector. This system was established in an H. pylori strain carrying a precise, unmarked chromosomal deletion of the vacA gene, which was constructed by adapting the streptomycin sensitivity system to H. pylori. It is based on the expression of the H. pylori rpsL gene as a counterselectable marker in the genetic background of an rpsL mutant. The utility of this approach is demonstrated by the expression of a recombinant gene encoding vacuolating cytotoxin (vacA) and a recombinant gene encoding an adherence-associated outer membrane protein (alpA) in H. pylori. Received: 10 May 1999 / Accepted: 7 July 1999     

Effect of outer sphere anions on the structure and color of nitrosylpentaamminechromium complex

Three kinds of crystalline compounds containing the nitrosylpentaamminechromium complexes [Cr(NO)(NH₃)₅]²⁺(A) were obtained: chloride ACl₂ (red-orange), chloride perchlorate ACl(ClO₄) (brown), and perchlorate A(ClO₄)₂ (green). The cause of the color change of the complex A with the change of outer sphere anions was sought using X-ray structural data of ACl₂, ACl(ClO₄), and A(ClO₄)₂. Crystal data: ACl₂, orthorhombic, space group Cmcm, a=10.0236 (9) Å, b=9.098 (3) Å, c=10.357(1) Å, V=944.5 (5) ų, Z=4; ACl(ClO₄), tetragonal, space group P4/nmm, a=7.6986 (8) Å, c=9.9566(8) Å, V=590.1 (1) Å^3,Z=2; A(ClO₄)₂, orthorhombic, space group Pnma, a=15.760 (2) Å, b=11.480(2) Å, c=7.920 (2) Å, V=1432.9 (4) ų, Z=4. The complex cation in ACl₂ has a distorted octahedral structure with a linear CrNO moiety. The short CrN (nitrosyl) distance of 1.692 (7) Å indicates the presence of multiple bonding between the chromium atom and the nitrogen atom in the nitrosyl group. The interatomic distances and angles within the complex cations hardly change with the change of the counter anions, while the distances between the complex cations in each crystal increase in the order ACl₂<ACl(ClO₄)<A(ClO₄)₂. The bulky perchlorate anions seems to separate the complex cations, while smaller chloride anions are not large enough to separate them. The distance (3.213(5) Å) between O(NO) and N(NH₃ in the adjacent complex cation) is rather short in the crystal of ACl₂, and there are six hydrogen bonds, where the NO group is surrounded by four NH₃ ligands. The distance (4.002(5) Å) between O(NO) and N(NH₃) is much longer in the crystal of A(ClO₄)₂, indicating the presence of no hydrogen bonding. In the crystal of ACl(ClO₄) the distance (3.452(4) Å) between O(NO) and N(NH₃) is in between those of ACl₂ and A(ClO₄)₂. The presence of hydrogen bonding between O(NO) and N(NH₃ in the adjacent complex cation) seems to cause the color change with the change of outer sphere anions.     

Conformation-dependent antibody response to Pseudomonas aeruginosa outer membrane proteins induced by immunization in humans

Outer membrane proteins (OMPs) of pathogenic bacteria have been used as protective antigens in developing bacterial vaccines. In the present study, we compared the antibody responses to a Pseudomonas aeruginosa OMP vaccine elicited in humans and rabbits by immunization. Immunization with the vaccine induced high titers of serum IgG antibody both in rabbits and humans but reactivities of the induced antibodies with the OMPs were different. The rabbit immune sera recognized most of the OMPs in the vaccine both in immunoblot and immunoprecipitation analyses. In contrast, a great variation in band pattern and intensity was observed among the human immune sera in immunoblot analysis, but not in immunoprecipitation analysis. Denaturation of the OMPs did not affect the binding activity of the rabbit immune sera as determined by ELISA, but substantially reduced those of the human immune sera and anti-OMP IgG purified from a pooled normal human plasma. These data suggest that antibody response to P. aeruginosa OMPs elicited by immunization in humans is mainly directed against discontinuous or conformation-dependent epitopes, which should be taken into account in developing vaccines, especially for OMP-derived synthetic peptides.     

Modulation of lymphocyte proliferative responses by a canine Lyme disease vaccine of recombinant outer surface protein A (OspA)

The modulation of human lymphocyte proliferative responses was demonstrated with a recombinant outer surface protein A (OspA) vaccine preparation for the prevention of Borrelia burgdorferi infection. After exposure to either the unaltered vaccine preparation or OspA prepared in saline, normal lymphocyte responses to the mitogens concanavalin A, phytohemagglutinin-M or pokeweed mitogen, or the antigen BCG were consistently reduced. Whole cell extracts of B. burgdorferi also modulated immune responses but required a much greater quantity of protein than needed for the OspA preparation. The magnitude of modulation was directly dependent on the quantity of OspA. OspA interferes with the response of lymphocytes to proliferative stimuli including a blocking of cell cycle phase progression. Future studies designed to delete the particular region or component of the OspA molecule responsible for this effect may lead to improved vaccine preparations.     

Mechanisms of Action of Rabbit CAP18 on Monolayers and Liposomes Made from Endotoxins or Phospholipids

We have investigated the mechanism of action of the cationic antimicrobial protein (18 kDa) CAP18 on liposomes and monolayers made from phospholipids and enterobacterial lipopolysaccharides (LPS). CAP18 intercalates into lipid matrices composed of LPS from sensitive strains, weaker into those made of LPS from a resistant strain (Proteus mirabilis strain R45) or negatively charged phospholipids, but not into those composed of neutral phosphatidylcholine. From the combination of data obtained with fluorescence resonance energy transfer and Fourier-transform infrared spectroscopy and film balance measurements, it can be concluded that structural differences in the LPS determine the depth of intercalation of CAP18 into the respective lipid matrices. Thus, we identified the L-Arap4N linked to the first Kdo of the LPS of P. mirabilis strain R45 to be responsible for the CAP18 resistance of this strain. These data provide insight into CAP18-mediated effects on the integrity of the outer membrane of Gram-negative bacteria and led to an improved model for rabbit CAP18 membrane interaction. Received: 14 January 2000/Revised: 20 April 2000     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram-negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non-ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.