Outer Membrane Preparations Research Articles

The electron microscopic cytochemical localization of arylsulphatase C activity in rat liver

Rat liver and most mammalian tissues possess three so-called arylsulphatase activities (arylsulphate sulphohydrolase, EC 3.1.6.1). Two of them, known as arylsulphatases A and B, are soluble lysosomal enzymes the function of which in vivo is to hydrolyse sulphated carbohydrate components of macromolecules. The third enzyme, known for many years as arylsulphatase C, is firmly bound to the endoplasmic reticulum membrane and is almost certainly a sulphatase active in vivo towards steroid arylsulphates (see Dodgson & Rose, 1975; Rose, 198%). This enzyme is therefore the only true mammalian arylsulphatase although it now seems that it may also be responsible for the well-recognized activity of the endoplasmic reticulum towards steroid alkylsulphates (Rose, 19823). The natural substrates are now recognized, but the enzymes are still routinely assayed with synthetic chromogenic arylsulphates, namely p-nitrocatechol sulphate for A and B and p-nitrophenyl sulphate or p-acetylphenyl sulphate for C. The enzymes, however, display some degree of cross-specificity towards these substrates and this has led to considerable confusion in enzyme identification, especially where the compounds have been used for their electron microscopic detection (Hopsu-Havu & Helminen, 1974). An absolutely specific assay for enzyme C using pacetylphenyl sulphate in phosphate buffer (which will inhibit enzymes A and B) had been developed (Milsom et al., 1972) and was therefore potentially useful in electron microscopy. The method, however, required modification because the use of phosphate precluded the use of heavy-metal trapping agents. The specific assay of enzyme C without relying on phosphate has been investigated (unpublished work) and can be accomplished with a high degree of accuracy by choosing suitable conditions. This has provided a basis for the specific detection of the enzyme in whole tissues and subcellular fractions. Studies on the distribution of enzyme C in rat liver using the method have confirmed the results of earlier cell fractionation work. Furthermore, the membrane localization of the enzyme is confirmed and from studies on the activity of pelleted microsomal fraction, activity is most likely located on or near the external surface (unpublished work). These observations supported the results of enzyme inhibition studies (Rose, 19823). Variable amounts of activity were also occasionally seen in the nuclear membrane (Fig. 1) and highly purified nuclear outer membrane preparations have been found to possess some arylsulphatase C activity (specific activity approx. 25% that of the endoplasmic reticulum). This had not been suspected previously (Milsom & Rose, 1970). The enzyme appears to be distributed throughout the cell but a pronounced deposition of product in particular regions of the endoplasmic reticulum, namely between clustered mitochondria in close proximity to the nucleus

Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody.

Hybridomas derived from mice immunized with Neisseria meningitidis serogroup B serotype 2b (B,2b) outer membrane preparations produced monoclonal antibodies (MAbs) specific for major outer membrane proteins of classes 1, 2, and 5. The MAbs were examined by enzyme-linked immunosorbent assay against a selected panel of seven strains of N. meningitidis (B,2b) of different sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, a serotype 2a, and a nontypable strain. The five MAbs selected were all bactericidal and of different immunoglobulin subclasses. None of the MAbs reacted with other bacterial strains in a dot-enzyme immunoassay. The corresponding antigenic determinant for each MAb was localized on a specific outer membrane protein by immunoblotting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of major outer membrane proteins. MAbs M5-11 and M5-30 bound to the class 2 protein and were serotype 2b specific. MAb M2-20 bound to the class 1 protein, and MAbs M5-16 and M5-19 bound to the class 5 protein. A mouse model of infection was established whereby a local infection progressed to lethal bacteremia over 3 days, and 50% of the animals were killed with an intraperitoneal injection of 10 meningococci plus 4% mucin and 1.6% hemoglobin. The ability of the MAbs to provide passive protection against experimental infection with N. meningitidis (B,2b) was examined. Both serotype-specific MAbs M5-11 and M5-30 were highly protective even though they were of different immunoglobulin subclasses. The class 5-specific MAb offered no protection, while the class 1-specific MAb gave limited protection. It may therefore be possible to provide protection against serotype 2b infection by using as vaccine the class 2 serotype-specific surface-exposed outer membrane protein epitopes defined by MAb M5-11 or M5-30.

Immunochemical characterization of the outer membrane complex of Serratia marcescens and identification of the antigens accessible to antibodies on the cell surface.

Serratia marcescens New CDC O14:H12 contains major outer membrane proteins of 43.5 kDal, 42 kDal (the porins) and 38 kDal (the OmpA protein) which can be separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Immunoblotting of whole cell or outer membrane preparations using antiserum raised against the whole cells revealed similar complex patterns of antigens. The OmpA protein was the major immunogen, although six other outer membrane proteins were also detected; the porins reacted only weakly with antibodies in this system. Immunoabsorption of antisera with whole cells showed that only the O antigenic chains of lipopolysaccharide and the H (flagella) antigens were accessible to antibody on the cell surface. Failure to detect the OmpA protein and other envelope antigens in this way suggests that their antigenic sites are not able to react with antibodies and are possibly masked by the O antigen.

Subtyping of Danish Haemophilus influenzae type B by their 45000 and 46000 molecular weight proteins.

In order to obtain a way of subtyping Haemophilus influenzae type b strains, outer-membrane protein patterns were investigated. Outer-membrane proteins from 45 different Haemophilus influenzae type b strains were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The non-heated sarkosyl insoluble outer-membrane preparations all contained four major proteins with molecular weights (Mr) of 39,000, 33,000, 30,000, and 28,000, except for one strain which possessed a protein weighing approximately 32,000. After heating the samples a 45,000 Mr protein appeared in 43 of the strains, and a 46,000 Mr in two. The 39,000 Mr protein remained unchanged, whereas the 33,000 Mr protein disappeared. The 30,000 and the 28,000 Mr proteins became more clearly separated. A faint 16,000 Mr band was observed in both heated and non-heated samples. 42 strains with the light heat-modified protein band had identical protein patterns and could not be subdivided by the methods used and may therefore constitute a clone. The remaining strains had individual patterns.

Effect of growth in serum on uptake of Neisseria gonorrhoeae by human neutrophils.

Because serum exposure precedes interaction of invasive Neisseria gonorrhoeae with neutrophils, neutrophil association with gonococci grown in serum was assessed. Uptake of a nonpiliated serum-resistant strain grown in 20% serum was reduced to 63.5% of control values. Heated serum (20%) yielded similar results. Stimulation of hexose monophosphate shunt activity in neutrophils by heated serum-grown gonococci (a phenomenon reflecting phagocytosis) was 64.5% of control values. Because gonococcal outer membrane (OM) structures mediate interaction with neutrophils, lithium acetate-treated OM preparations of gonococci grown in heated serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Multiple new protein bands and a quantitative decrease in amounts of proteins I, II, and III were noted. However, a decrease in the amounts of these membrane proteins was not observed with alternative membrane extraction techniques. Gonococcal growth in 20% serum dialyzed in 3,500-molecular-weight exclusion tubing allowed normal neutrophil association and OM protein expression.

Pollen Sporoplasts: Dissolution of Pollen Walls

4-Methylmorpholine N-oxide monohydrate (MMNO.H(2)O), a potent solvent for polysaccharides, is an effective vehicle for release of membrane-enclosed male gametophytes (sporoplasts) from spore walls. This release occurs in minutes when pollen (Lilium longiflorum Thunb.) is suspended in a melt of MMNO.H(2)O at 75 degrees C. Continued heating at 75 degrees C leads to distintegration of the exine ;shell' which coalesces into immiscible globules in the MMNO melt. These observations provide a general procedure for preparation of pollen sporoplasts and sporoplast outer membranes, and offer a new method for dissolving the sporopollenin component of the spore wall.

Pili of Neisseria meningitidis. Analysis of structure and investigation of structural and antigenic relationships to gonococcal pili.

To provide information useful for the design of a pilus vaccine effective for the prevention of both meningococcal and gonococcal disease, the electron microscopic morphology of meningococcal pili and the structural and antigenic relationships of meningococcal pili to gonococcal pili were investigated. Meningococcal pili were 4-6 nm in width, extended 500-6,000 nm from the organism surface, and occurred singly or in bundles composed of 8-10 pili per bundle. Meningococcal pilin varied between 17,250 and 20,600 daltons. Pilin was present in outer membrane preparations of some meningococcal isolates that were nonpiliated by electron microscopic examination. Antibodies to gonococcal pili, cyanogen bromide cleavage fragments of gonococcal pilin, or synthetic peptide analogues corresponding to regions of the gonococcal pilin sequence, were used to detect common meningococcal and gonococcal antigenic determinants that might indicate the existence of a conserved sequence beyond residue 29. Antibody to intact gonococcal pili or to the variable CNBR-3 region of gonococcal pilin detected little shared antigenicity with meningococcal pilin. However, pilin from all tested meningococcal isolates reacted with antibody to the CNBR-2 fragment of gonococcal pilin, a region highly conserved among gonococcal strains. Meningococcal pilins were also broadly crossreactive with antibody to a synthetic peptide corresponding to residues 69-84 of the gonococcal sequence, a part of the CNBR-2 region that appears to be critical for gonococcal receptor-binding function. If a sequence similar to 69-84 is also important for receptor-binding function in meningococcal pili, a peptide corresponding to this region may elicit antibodies that block the adherence function of pili elaborated by both Neisseria gonorrhoeae and N. meningitidis.

Identification and characterization of Vibrio cholerae surface proteins by radioiodination.

Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides.

Disulfide-bonded outer membrane proteins in the genus Legionella.

Legionella pneumophila and related species were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for outer membrane proteins. Of the 10 species examined, 9 contained a 24-kilodalton (kDa) major outer membrane protein (MOMP) that was resolvable only when outer membrane material was heated in the presence of 2-mercaptoethanol. Labeling studies with [35S]cysteine indicated that the protein contained cysteine, and disulfide cross-linking of the unreduced complex was demonstrated by labeling with iodoacetamide. The unreduced outer membrane preparation contained peptidoglycan, and after treatment with lysozyme to remove peptidoglycan, a protein complex of 95 kDa was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Reduction of the 95-kDa complex yielded 24-kDa monomers, suggesting that the 95-kDa complex was composed of four subunits. The 24-kDa MOMP from L. pneumophila was purified, and antibody produced to this protein cross-reacted with all species of Legionella as determined from an immunoblot of a sodium dodecyl sulfate gel. Only serogroup 1 strains of L. bozemanii lacked the 24-kDa MOMP and showed no cross-reactivity. These results suggest that the 24-kDa MOMP common to most species of Legionella contains a genus-specific epitope.

High-level synthesis of the phage lambda outer-membrane protein from the cloned lom gene

A 2.7-kb Kpn I- EcoRI fragment carrying the lom gene of bacteriophage λ has been cloned into plasmid pPR42 and recloned into the SmaI site of pUC9. Large quantities of Lorn were seen in outer-membrane (OM) preparations of strains carrying the latter clone and its derivatives. The reading frame of lom was identified as ORF206a. The protein was not demonstrably associated either covalently or non-covalently with the peptidoglycan layer of the cell envelope.

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

Immunoblot method for identifying surface components, determining their cross-reactivity, and investigating cell topology: Results with Haemophilus influenzae type b

Methods currently used for identifying exposed membrane components of gram-negative bacteria can give false positive results, and also do not provide information on nonprotein components such as lipopolysaccharide and polysaccharide capsules. A method, described within, has been developed to overcome these limitations. Briefly an outer membrane preparation derived from encapsulated (type b) Haemophilus influenzae was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated components were then transferred electrophoretically to nitrocellulose. The nitrocellulose was cut into vertical strips, which were then each incubated with rabbit antiserum to the whole bacterium or with the same antiserum after absorption with any of the following: the same strain of H. influenzae, a capsule-deficient mutant of that strain, other strains of H. influenzae, or other bacteria. The strips were then incubated with 125I-protein A, and the bound antibodies were detected by autoradiography. The autoradiograph of the strip exposed to unabsorbed antisera revealed the identity of those individual outer membrane components that bound antibodies. A comparison of the intensity of the various bands on this strip with those on the strips exposed to absorbed antisera was then used to identify (1) surface-exposed components, (2) those components occluded by capsule, and (3) cross-reactivity of exposed components. This method should be applicable to other cells and subcellular particles. Its major disadvantage is that it can provide false negative results.

Haemophilus influenzae type b isolates show antigenic variation in a major outer membrane protein

Antigenic variation of the outer membrane proteins among isolates of Haemophilus influenzae was examined by immunoblotting. Rabbit antisera were raised against six strains of H. influenzae type b and tested against outer membrane preparations of 50 isolates. The principal outer membrane band was not reactive on immunoblotting, so its antigenic heterogeneity could not be examined. Most of the other outer membrane proteins shared common determinants among all strains tested. Absorption of serum with heterologous bacteria removed antibody to nearly all proteins, confirming the extensive cross-reactivity among isolates. The greatest antigenic variation was seen in one major outer membrane band, a heat-modifiable, Zwittergent-soluble protein with a molecular weight of 49,000 to 51,000. One antiserum reacted with the 49,000-to-51,000-molecular-weight protein of the homologous isolate only; the remaining five antisera showed differing patterns of reactivity with heterologous 49,000-to-51,000-molecular-weight proteins. We were able to divide the 50 H. influenzae isolates into 13 antigenic groups based on their reaction patterns. The antigenic groupings may provide an epidemiological tool for studying the prevalence and transmission of strains of H. influenzae type b.

Loss of pili and decreased attachment to human cells by Neisseria meningitidis and Neisseria gonorrhoeae exposed to subinhibitory concentrations of antibiotics.

Recent evidence has suggested that surface structures of pathogenic bacteria, which are important in attachment to human mucosal surfaces, may be absent on bacteria grown in the presence of subinhibitory concentrations of antibiotics. We studied the effect of tetracycline and penicillin on meningococcal and gonococcal pili. Subinhibitory concentrations of tetracycline and penicillin were found to markedly reduce the number of pili per meningococcus or gonococcus and the percentage of meningococci or gonococci with pili, as determined by negative-staining electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane preparations suggested that tetracycline decreased expression of pili by inhibiting synthesis of pilin subunits. In contrast, pilin subunit synthesis was unaltered by penicillin, suggesting a defect in assembly of pilin subunits or in anchoring of assembled pili. The decrease in the number of pili that occurred with subinhibitory concentrations of both tetracycline and penicillin was accompanied by a marked decrease in the ability of the organisms to attach to human cells. Gonococci or meningococci removed from the influence of subinhibitory concentrations of the antibiotics regained piliation, and attachment returned to levels near those of controls. The expression of meningococcal and gonococcal pili may be affected by factors that influence synthesis of pilin subunits or factors that interfere with the assembly and anchoring of pili in the outer membrane.

Strain and temperature-dependent variation in the amount of BtuB polypeptide in theEscherichia coliK-12 outer membrane

The outer membrane protein BtuB of Escherichia coli K-12 is the receptor for vitamin B12; it is normally present in approx. 200 copies per cell. We describe here the conditions by which BtuB was readily observed in electropherograms of outer membrane preparations. These conditions are as follows. (1) Incorporation of 8 M urea in sodium dodecyl sulfate-polyacrylamide gel systems improved detection of the polypeptide. (2) In most E. coli K-12 strains examined, BtuB was most abundant when cells were grown at 27°C. This thermoregulation of BtuB was independent of envZ and envY, two regulatory genes for outer membrane proteins.

Serum sensitivity of a Pseudomonas aeruginosa mucoid strain.

The susceptibility of Pseudomonas aeruginosa 144M (a mucoid strain isolated from the sputum of a cystic fibrosis patient) to the bactericidal activity of pooled fresh normal human serum (FHS) was examined. FHS at concentrations of greater than or equal to 2.5% was capable of killing greater than 95% of strain 144M. Strain 144M was killed by FHS in a dose-dependent manner. Although either immunoglobulin M (IgM) or IgG was bactericidal in the presence of complement, IgM was about 10 times as effective as IgG. However, optimal killing activity required both IgM and IgG and complement, activated by the classical pathway. A role for lysozyme in the killing of 144M was demonstrated only when low concentrations of FHS were used. In contrast to 144M, P. aeruginosa strains 144NM and 144M(SR) were totally resistant to FHS at all of the concentrations tested (up to 50%). Neither the FHS susceptibility of 144M nor the FHS resistance of 144NM or 144M(SR) was altered by choice of growth medium, growth phase, or temperature of growth. Results of absorption studies with whole organisms, isolated outer membrane preparations, or lipopolysaccharide (LPS) from each strain suggest that the antigen(s) which binds the bactericidal immunoglobulins is accessible on the surface of 144M but not on the surface of 144NM or 144M(SR), is insensitive to trypsin treatment, and is believed to be LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the three LPS preparations demonstrated that 144M LPS contained primarily lipid-A-core polysaccharide components, whereas the LPS from 144NM and 144M(SR) were heterogeneous, with various degrees of O-side-chain substitution. These results suggest that at least one target for bactericidal antibody on the surface of 144M is contained in the rough LPS of this strain.

Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis.

An outer membrane complex containing hemagglutinating and bacterial aggregating activity has been isolated from Bacteroides gingivalis. Examination of the membrane material by biochemical analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological means revealed that the crude outer membrane preparation contained three major proteins and a lipopolysaccharide population that displayed size heterogeneity. At least two membrane proteins as well as the lipopolysaccharide were found to be antigenically active by immunoblot analysis. With gel chromatography and a lipopolysaccharide disaggregating buffer the membrane material was separated into two fractions. An accompanying separation of the two adherence activities was observed. The first membrane fraction, containing mostly protein and carbohydrate material, was found to contain the bacterial aggregating activity. This fraction also contained a high-molecular-weight lipopolysaccharide population. The second membrane fraction, consisting of low-molecular-weight lipopolysaccharide, protein, and loosely bound lipid was found to contain the hemagglutinating activity.

Evaluation of the human immune response to outer membrane proteins of Vibrio cholerae.

The immune response of 114 volunteers with diarrhea after experimental challenge with four strains of Vibrio cholerae O1 was characterized in a microtiter enzyme-linked immunosorbent assay antibody detection system by using a partially purified outer membrane preparation (OMP) from these strains as an antigen. Analysis of paired sera from 29 persons with noncholera diarrhea (negative control population), demonstrated that a rise in net optical density greater than 0.10 was significant. A total of 50% of the 79 cholera volunteers challenged with El Tor biotype and 54% of the 35 volunteers challenged with classical biotype had significant rises in immunoglobulin G anti-OMP. Paired sera that showed significant rises when tested against homologous OMP all manifested significant rises when also tested against a serotype-heterologous OMP. Immunoblotting techniques showed that the antigens to which antibodies were reacting were mainly protein in nature, not lipopolysaccharide. Furthermore, absorption with lipopolysaccharide decreased the optical density by a mean of only 12% (0 to 30%), corroborating that antibody was mainly directed against OMP and not lipopolysaccharide. This study indicates that there is a human immunoglobulin G response to OMP during clinical cholera infection and that this response is constant among bio- and serotypes.

Evidence for the participation of N-acetylated amino sugars in the coaggregation between Cytophaga species strain DR2001 and Actinomyces israelii PK16.

Coaggregation between Cytophaga sp. strain DR2001 and Actinomyces israelii PK16 was partially inhibited by certain N-acetylated amino sugars (N-acetylneuraminic acid, N-acetylgalactosamine, and N-acetylglucosamine) and was completely inhibited by the trisaccharide neuraminin-lactose. The monosaccharides exerted their effect at concentrations between 30 to 100 mM, whereas the trisaccharide was an effective inhibitor at significantly lower concentrations. Outer membrane preparations caused A. israelii PK16 cells to aggregate; however, vesicles released from the cell envelope during growth failed to do so. Adherence studies with a non-coaggregating mutant of the cytophaga suggest that the spheroidal hydroxyapatite attachment sites and coaggregation receptors are separate entities.

The identification of outer membrane proteins and flagella of Campylobacter jejuni.

The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.